Amicon Ultra-4 Centrifugal Filter Devices User Guide

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milipure超滤管说明书

EnglishIntroductionAmicon® Ultra-15 10K centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors and easy concentrate recovery from dilute and complex sample matrices. Thevertical design and available membrane surface area provide fast sample processing, high sample recovery (typically greater than 90% of dilute starting solution), and the capability for 80-fold concentration. Typical processing time is 15 to 40 minutes. Solute polarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the filter device prevents spinning todryness and potential sample loss. The concentrate is collected from the filter device sample reservoir using a pipettor, while the ultrafiltrate is collected in the provided centrifuge tube. The device can be spun in a swinging-bucket or fixed-angle rotor. Amicon® Ultra-15 10K devices are supplied non-sterile and are for single use only.Intended UseThe Amicon® Ultra-15 product line includes 5 different cutoffs (Molecular Weight Cutoff, MWCO), however, the Amicon® Ultra-15 10K device (10,000 MWCO) is the only device intended for in vitro diagnostic use. It can be used to concentrate serum, urine, cerebrospinal fluid, and other body fluids prior to analysis. For information on other Amicon® Ultra-15 cutoffs, go to and enter Amicon Ultra-15in the search box.User GuideAmicon ® Ultra-15 10K Centrifugal Filter Devicesfor volumes up to 15 mLUFC901008UFC901024UFC901096CVApplications●Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids(DNA/RNA samples, either single- or double-stranded), microorganisms, column eluates, andpurified samples●Purification of macromolecular components found in tissue culture extracts and cell lysates, removalof primer, linkers, or molecular labels from a reaction mix, and protein removal prior to HPLC●Desalting, buffer exchange, or diafiltrationMaterials SuppliedThe Amicon® Ultra-15 10K device is supplied with a cap, a filter device, and a centrifuge tube.Required Equipment●Centrifuge with swinging-bucket or fixed-angle rotor with wells/carriers that can accommodate50 mL tubesCAUTION: To avoid damage to the device during centrifugation, check clearance before spinning.●Pipettor with 200 microliter (μL) tip for concentrate recoverySuitabilityPreliminary recovery and retention studies are suggested to ensure suitability for intended use. See the “How to Quantify Recoveries” section.Shelf LifeShelf life is 3 years from date of manufacture. Refer to expiration date on package label.Rinsing Before UseThe ultrafiltration membranes in Amicon® Ultra-15 10K devices contain trace amounts of glycerine. If this material interferes with analysis, rinse the device with buffer or Milli-Q® water before use. If interference continues, rinse with 0.1 N NaOH followed by a second spin of buffer or Milli-Q® water. CAUTION: Do not allow the membrane in Amicon® Ultra filter devices to dry out once wet. If you are not using the device immediately after rinsing, leave fluid on the membrane until the device is used. How to Use Amicon® Ultra-15 Centrifugal Filter Devices1. Add up to 15 mL of sample (12 mL if using a fixed-angle rotor) to the Amicon® Ultra filter device.2. Place capped filter device into centrifuge rotor; counterbalance with a similar device.3. When using a swinging-bucket rotor, s pin the device at 4,000 × g maximum for approximately15–40 minutes.When using a fixed-angle rotor, orient the device with the membrane panel facing up and spin at 5,000 × g maximum for approximately 15–40 minutes.NOTE: Refer to Figure 1 and Table 1 for typical spin times.4. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdrawthe sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation. Desalting or DiafiltrationDesalting, buffer exchange, or diafiltration are important methods for removing salts or solvents in solutions containing biomolecules. The removal of salts or the exchange of buffers can be accomplished in the Amicon® Ultra-15 device by concentrating the sample, then reconstituting the concentrate to the original sample volume with any d esired solvent. The process of “washing out” can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced. See example below.1 mg/mL100 mMNaCl200 μL of75 mg/mLprotein inNaCl200 μL of75 mg/mLprotein inNaClAdd 14.8 mL of10 mM NaClor exchangebufferPerformanceFlow RateFactors affecting flow rate include sample concentration, starting vol u me, chemical nature of solute, relative centrifugal force, centrifuge rotor angle, membrane type, and temperature. Figure 1 and Table 1 can be used to estimate the time required to achieve a given volume of filtrate or concentrate. A typical spin time for a 15 mL sample is approximately 15 to 40 minutes. While most of the sample is filtered in the first 15 to 30 minutes of centrifugation, the lowest concentrate volume (100–150 μL) is reached after spinning for 15 to 40 minutes.Spin conditions: Swinging-bucket rotor (4,000 × g, 15 mL starting volume),or fixed-angle rotor, (5,000 × g, 12 mL starting volume), room temperature.Protein marker used: Cytochrome c, n=6.Table 1. Typical Concentrate Volume vs. Spin TimeSpin conditions: Room temperature.Protein marker used: Cytochrome c, n=6 (mean value of 3 membrane lots).Shaded volumes were used for the calculation of protein recovery in Table 3.Protein Retention and Concentrate RecoveryThe membranes used in Amicon® Ultra devices are characterized by a molecular weight cutoff (MWCO); that is, their ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the MWCO may be only partially retained. Membrane retention depends on the solute’s molecular size and shape. For most applications, molecular weight is a convenient parameter to use in assessing retention characteristics. For best results, use a membrane with a MWCO at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. Refer to Table 2.Table 2. Typical Retention of Protein MarkersMarker/Concentration MolecularWeightDeviceMWCO% RetentionSwinging-bucket% RetentionFixed-angleSpin Time(min)α-Chymotrypsinogen (1 mg/mL)25,00010K> 95> 9530 Cytochrome c (0.25 mg/mL)12,400> 95> 9530 Vitamin B-12 (0.2 mg/mL)1,350< 5< 530 Spin Conditions: Swinging-bucket rotor (4,000 × g, 15 mL starting volume), or fixed-angle rotor, (5,000 × g,12 mL starting volume), room temperature, n=6 (mean value of 3 membrane lots).Factors that determine sample recovery include the nature of the protein solute relative to the device MWCO chosen, starting concentration, and concentration factor. Table 3 provides typical recoveries for Amicon® Ultra-15 10K device.Table 3. Typical Concentrate RecoveryConcentrate Concentration ConcentrateSpin Conditions: Swinging-bucket rotor (4,000 × g, 15 mL starting volume), or fixed-angle rotor, (5,000 × g,12 mL starting volume), room temperature, n=6 (mean value of 3 membrane lots). The shaded volumes were taken from Table 1.Maximizing Sample RecoveryLow sample recovery in the concentrate may be due to adsorptive losses, over-concentration, or passage of sample through the membrane.●Adsorptive losses depend upon solute concentration, its hydrophobic nature, temperature and timeof contact with filter device surfaces, sample composition, and pH. To minimize losses, removeconcentrated samples immediately after centrifugal spin.●If the starting sample concentration is high, monitor the centrifugation process in order to avoid over-concentration of the sample. Over-concentration can lead to precipitation and potential sample loss.●If the sample appears to be passing through the membrane, choose a lower MWCO Amicon® Ultra-15device.How to Quantify RecoveriesCalculate total recovery, percent concentrate recovery, and percent filtrate recovery using the method below. The procedure provides a close approximation of recoveries for solutions having concentrations up to roughly 20 mg/mL.NOTE: Appropriate assay techniques include absorption spectrophotometry, radioimmunoassay, refractive index, and conductivity.Direct Weighing ProcedureThe density of most dilute proteins is nearly equal to the density of water (i.e., 1 g/mL). Using this property, the concentrate and filtrate volumes can be quantified by weighing them and converting the units from grams to milliliters. This technique is valid only for solutions with concentrations of approximately20 mg/mL or less.1. Before use, separately weigh the empty filter device, the centrifuge tube, and an empty tube forconcentrate collection.2. Fill filter device with solution and reweigh.3. Assemble device and centrifuge per instructions.4. Collect the concentrate with a pipettor and dispense it into the preweighed concentrate collection tube.5. Remove the device from the centrifuge tube and weigh the centrifuge tube and concentrate collectiontube.6. Subtract weight of empty device/tubes to calculate weights of starting material, filtrate, andconcentrate.7. Assay the starting material, filtrate, and concentrate to determine solute concentration.8. Calculate recoveries using the weight/volume data and the measured concentrations as follows:% concentrate recovery = 100 × W c × C c W o × C o% filtrate recovery = 100 × W f × C f W o × C o% total recovery = % concentrate recovery + % filtrate recoveryW c = total weight of concentrate before assayW o = weight of original starting materialW f= weight of filtrateC c= concentrate concentrationC o= original starting material concentrationC f= filtrate concentrationSpecificationsMaximum initial sample volumeSwinging-bucket15.0 mLFixed-angle rotor12.0 mLTypical final concentrate volume 150–300 μLMaximum relative centrifugal forceSwinging-bucket rotor4,000 × gFixed-angle rotor5,000 × gActive membrane area7.6 cm2DimensionsFilter device in tube (capped)Length: 119 mm (4.7 in.)Diameter: 33.5 mm (1.3 in.) Filter deviceLength: 72.0 mm (2.8 in.)Diameter: 29.7 mm (1.2 in.) Materials of ConstructionFilter device Copolymer styrene/butadieneMembrane Ultracel® low binding regenerated cellulose Filtrate tube PolypropyleneFiltrate cap and liner PolyethyleneChemical CompatibilityAmicon® Ultra centrifugal devices are intended for use with biological fluids and aqueous solutions. Before use, check the sample for chemical compatibility with the device.Table 4. Chemical Compatibility of Amicon® Ultra Filter DevicesAcids Concentration Concentration Acetic acid ≤ 50%*Phosphoric acid ≤ 30% Formic acid ≤ 5%*Sulfamic acid≤ 3% Hydrochloric acid ≤ 1.0 M Sulfuric acid ≤ 3% Lactic acid ≤ 50%Trichloroacetic acid (TCA)≤ 10%* Nitric acid ≤ 10%Trifluoroacetic acid (TFA)≤ 30%* AlkalisAmmonium hydroxide ≤ 10%Sodium hydroxide ≤ 0.5 M Alcoholsn-Butanol ≤ 70%Isopropanol ≤ 70% Ethanol ≤ 70%Methanol ≤ 60% DetergentsAlconox® detergent ≤ 1%Sodium dodecyl sulfate (SDS)≤ 0.1% CHAPS detergent≤ 0.1%Tergazyme®detergent ≤ 1% Lubrol® PX detergent≤ 0.1%Triton® X-100 surfactant≤ 0.1% Nonidet™ P-40 surfactant≤ 2%Tween® 20 surfactant≤ 0.1% Sodium deoxycholate≤ 5%Organic solventsAcetone not recommended Ethyl acetate not recommended Acetonitrile ≤ 20%Formaldehyde ≤ 5% Benzene not recommended Pyridine not recommended Carbon tetrachloride not recommended Tetrahydrofuran not recommended Chloroform not recommended Toluene not recommended Dimethyl sulfoxide (DMSO)≤ 5%*MiscellaneousAmmonium sulfate Saturated Phenol≤ 1% Diethyl pyrocarbonate ≤ 0.2%Phosphate buffer (pH 8.2)≤ 1 M Dithiothreitol (DTT)≤ 0.1 M Polyethylene glycol≤ 10% Glycerine≤ 70%Sodium carbonate ≤ 20% Guanidine HCl ≤ 6 M Tris buffer (pH 8.2)≤ 1 M Imidazole≤ 100 mM Urea ≤ 8 M Mercaptoethanol≤ 0.1 M* Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are recommended to determine whether leachables represent potential assay interferences.Product Labeling SymbolsThe following table defines the symbols found on Amicon® Ultra-15 10K device labels.Product Ordering InformationThis section lists the catalogue numbers for Amicon® Ultra Ultrafiltration Devices. See the Technical Assistance section for contact information. You can purchase these products on-line at/products.* Amicon® Ultra-4 and -15 10K devices are for in vitrodiagnostic use. All other devices are for research use only.NoticeThe information in this document is subject to change without notice and should not be construed as a commitment by Merck Millipore Ltd. (“Millipore”) or an affiliate. Neither Merck Millipore Ltd. nor any of its affiliates assumes responsibility for any errors that may appear in this document.Technical AssistanceFor more information, contact the office nearest you. Up-to-date world-wide contact information is available on our web site at /offices. You can also visit the tech service page on our web site at /techservice.Standard WarrantyThe applicable warranty for the products listed in this publication may be found at/terms (“Conditions of Sale”).M Merck Millipore Ltd.Tullagreen,Carrigtwohill,Co. Cork, IrelandMade in IrelandThe M logo, Millipore, Amicon, Milli-Q, and Ultracel are registered trademarks of Merck KGaA, Darmstadt, Germany.All trademarks of third parties are the property of their respective owners.© 2014 EMD Millipore Corporation. Billerica, MA, U.S.A. All rights reserved.PR04318TR, Rev. B, English, 10/14。

碧云天 L31600 pLenti-TLR2-sgRNA 产品说明书

pLenti-TLR2-sgRNA产品简介：pLenti-TLR2-sgRNA (TLR2基因敲除质粒)是一种在动物细胞中可以同时表达Cas9、目的基因的sgRNA 和puromycin 抗性基因的质粒。

用于在动物细胞中直接基于CRISPR/Cas9技术敲除目的基因，或者通过包装慢病毒后基于CRISPR/Cas9技术敲除目的基因。

本质粒中sgRNA 的有效性已经通过T7EI 法的验证。

本质粒在细菌中为Amp 抗性，全长约13,000bp 。

本质粒的关键图谱信息请参考图1。

本质粒可直接转染细胞用于目的基因的CRISPR/Cas9敲除，以及通过puromycin 筛选稳定细胞株；也可以与pMDLg 、Rev 及VSV-g 共转HEK293T 细胞进行重组慢病毒(lentivirus)的包装，然后再用于感染细胞或组织并进行目的基因的CRISPR/Cas9敲除。

图1. 表达sgRNA 、Cas9和puromycin 抗性的pLenti-sgRNA 质粒关键图谱信息。

本质粒中的sgRNA 基于碧云天研发的CRISPR/Cas9 sgRNA 快速筛选和验证体系获得，sgRNA 的有效性已经通过T7EI 法验证。

本质粒用于实验时，建议同时选购无任何靶向的对照质粒pLenti-Control-sgRNA (L00011)或靶向GFP 的对照质粒pLenti-GFP-sgRNA (L00013)。

碧云天同时提供基于CRISPR/Cas9技术的TLR2基因敲除的质粒(L31600 pLenti-TLR2-sgRNA)、慢病毒(L31601 TLR2 Knockout Lentivirus)、HEK293T 细胞(L31602 TLR2 Knockout HEK293T Cells)、HEK293T 敲除细胞的RIPA 裂解液(L31603 TLR2 Knockout HEK293T RIPA Lysate)、HEK293T 敲除细胞的Trizol 裂解液(L31604 TLR2 Knockout HEK293T Trizol Lysate)等产品，具体请在碧云天网站查询或在本产品网页点击相应产品。

Agilent-GCMS培训(完整版330页)

炉箱排气出口
进样口和检测器出口
气路连接口
电缆连接口
电源连接口
炉箱冷却风进口
Agilent 7890A 键盘介绍
*
运行按键
气相部件按键
数字按键
信息按键
方法存储和自动运行按键
维护按键
*
Agilent GC6890前视图
*
Agilent GC6890侧后视图
*
即时功能键功能键快捷键信息键数字键多功能键方法存贮与自动运行
MSD
10-5 Torr
<2 mL/min
760 Torr
0.5 - 15 mL/min
传输线
*
气相色谱和质谱的联用技术——传输线
传输线
涡轮泵
自动进样器
离子源
炉箱
*
色谱柱
流量控制器
稳压器
空气
氢气
载气
分子筛
脱水管
固定
进样口
检测器
电子部件
PC
限流器
典型的气相色谱
*
分子筛 P/N 5060-9084
(0 to 218 V)
Signal Out
EM Voltage
电子倍增器
电压有使用上限（3000伏）电压的提高，可以提高检测器的信号
*
电子倍增器的寿命曲线
*
提供足够的平均自由程提供无碰撞的离子轨道减少离子-分子反应减少背景干扰延长灯丝寿命消除放电增加灵敏度
为什么MS需要真空
空气压力应为 80 psi。
推荐管线压力
氢气压力应为 60 psi。
载气必须通过控制形成恒定的压力和恒定的流量。上下游控制器压差保持1公斤以上。

5 高分子分子量与尺寸

Mz > Mw > Mv > Mn
Molecular weight distribution
分子量分布
Number-average distribution width
M M n
2 n

2

n
Mw M M 1 n
2 n
Weight-average distribution width
a
c b
样品a：可纺性很差；样品b：有所改善；
5 10 15 M×10-4
样品c：由于分子量15～20万的大分子所占的比例较大，可纺性很好。
Average molecular weight 平均分子量
Molecular weight
Number Weight for each type of chain
1 a i

1/ a
Example:
ni
nM n
i i
10
10 20
10 10
Mi(×10-4) 30
Mn
i
10 30 10 20 10 10 104 20 104 10 10 10
10 302 10 202 10 102 Mw 104 23.3 104 ni M i 10 30 10 20 10 10
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超滤离心管的使用

•Used for Protein Purification and Concentration•Pack of 96 filter units and corresponding 2 mL centrifuge tubes•Filter holds 0.5 mL volume (we work with such small volumes the 2, 4, and 15 mL versions are excessive, thought could be used to wash faster/ more thoroughly if you so choose) •Filter has a 100 kDa NMWL membrane (MW cut-off of 100 kDa)Rough Protocol for Buffer Exchange:1.Assemble filter column as shown in the manufacturer's manual/protocol (see User Guide).装配离心管（两个管套在一起，外管是普通离心管，内管为过滤管）2.Add volume (~5-20 uL depending on number of samples being prepared for TEM) ofconcentrated ferritin in TBS to filter unit.取5-20 μL（根据TEM样品量选择具体的量）分散铁蛋白的TBS溶液，加入内管（过滤管）3.Fill remaining filter volume (to 500 uL) with DI/ultra-purified water.加入500 μL超纯水。

4.Spin at 14,000 x g for 10-15 minutes. 内管（过滤管）套在外管内，装入离心机，14000 x g离心力下离心10-15分钟。

Thermo Scientific Heraeus Megafuge 8和8R小型实验室离心机产品介

Thermo ScientiﬁcHeraeus Megafuge 8 Centrifuge SeriesFits in.Stands out.Introducing the NEW!Thermo Scientiﬁc™Heraeus™ Megafuge™ 8 and 8R small benchtop centrifugesFits in.4t o your space, with its compact footprint.Auto-Lock ™ Rotor ExchangeSecure push-button application versatility and cleaning convenienceClickSeal ™ Biocontainment LidsOne-handed, certiﬁed 1 sample protectionMaximized Swing-Out CapacityStands out.4d esigned for solid reliability and consistent results.14Ventilated (left) and refrigerated (right)n B iocontainment sealing options, including certiﬁed ClickSeal lids for glove-friendly, one-handed operation n M anufactured with quality materials providing broad Simple solutions.Flexible processing.Reliable quality. Safe operation.1B Public Health England, Porton Down, UK Fits in. Stands out.Secure, push-button Auto-Lock rotor exchange in as little as 3 seconds delivers:n Trouble-free rotor installation and removal linical processing up to 24 x 5/7 mL blood tubes per runAccommodates up to 30 spin columns, such as DNA and RNA mini-preps, Micro-volume Solution RotorsSample-ready.User-friendly.n O ne-touch operation with pre-saved protocolsn H ighly visible backlit display for easy reading of parameters across the labn O ptional indicators at end of run, including automatic lid opening, full ﬂashing display andadjustable audible signaln Glove- and detergent-friendlyn M ultilingual instructions – English, Dutch, French, German, Italian, Russian and Spanish — onprogramming, run conditions, alerts andservice messagesExpanded Programming Optionsn A lphanumeric program naming ,of up to 12 characters, promotes correct program selection every time.n P rogram-only mode limits thecontrol of the centrifuge to the set program and the control quadrant (Start, Stop, Pulse and Open), ideal for controlled environmentsFits in. CLINICAL APPLICATIONS.Outstanding capacity in a compact design with a smart, simple interface.n R uns up to 24 x 5/7 mL blood tubes at a time in swinging bucket conﬁguration n A uto-Lock rotor exchange simpliﬁes cleaning n C onforms to the latest clinical and safety standards n F ast acceleration and deceleration times for quicker spinsStands out.TX-150 Swinging Bucket RotorM10 Swinging Bucket Rotor8 x 50 mL Individually Sealed Fixed Angle RotorCLINIConic Fixed Angle RotorHematocrit Rotor1B iocontainment certiﬁcation by the Public Health England, Porton Down, UKDedicated Rotors for Routine Clinical ProcessingTX-100S Clinical Swinging Bucket Rotor with Sealed Carriers TX-100 Clinical Swinging Bucket Rotor with CarriersInnovative design supports multiple users and applications to stay one step ahead of your evolving research needs.n 8 x 50 mL swinging bucket rotor capacity, 4 microplates, or 30 ﬁltration columns for research needs n A uto-Lock rotor exchange for application ﬂexibility – go from 50 mL tube to microplates to microtubes effortlesslyFits in. RESEARCH APPLICATIONS.Stands out.to 30,279 x g enables • quicker spins• improved separations• access to a wider range of applications while maintaining full swing-out rotor fl exibility, combined with quick, safe Auto-Lock rotor exchange.TX-150 Swinging Bucket RotorM10 Swinging Bucket RotorMT-12 Swinging Bucket RotorHIGHConic III Fixed Angle RotorCLINIConic Fixed Angle RotorMicroClick 18 x 5 RotorMicro-Volume Solution RotorsMicroClick 24 x 2Fixed Angle Rotor8 x 8 PCR Strip FixedAngle Rotor1B iocontainment certiﬁcation by the Public Health England, Porton Down, UKTX-150 Swinging M10 SwingingBucket Rotor MT-12 Swinging Bucket Rotor23468Versatile Swinging Bucket RotorsMax Tube Max Speed Max RCFTX-100S Clinical Swinging Bucket Rotor TX-100 Clinical Swinging Bucket RotorClinical Swinging Bucket RotorsCat. No. DescriptionRotorCapacity(places xvolume, mL)Max TubeDimensions(O x L, mm)Max Speed(rpm)Max RCF(x g)Max Speed(rpm)Max RCF(x g)Clinical Swinging Bucket Rotors VENTILATED REFRIGERATED75005704TX-100S Clinical Swinging Bucket Rotorwith Sealed Carriers, 90°, R max 144 mm8 x 1517 x 1064,5003,2604,500 3,26075005705TX-100 Clinical Swinging Bucket Rotorwith Carriers, 90°, R max 144 mm16 x 1517 x 1214,5003,2604,5003,260Adapters for TX-100S Clinical Rotor and TX-100 Clinical Rotor (each)1120366613.5 mL Urine Tube16/8 x 13.516 x 82HIGHConic III Rotor8 x 50 mL Rotor MicroClick 18 x 5 Rotor8 x 8 PCR Strip RotorMicroClick 24 x 2 RotorMicroClick 30 x 2 RotorCat. No. Description Rotor Capacity(places xvolume, mL)Max TubeDimensions(O x L, mm)Max Speed(rpm)Hematocrit RotorThermo Scientiﬁc Heraeus Megafuge 8 Centrifuge SeriesSpeciﬁcationsSwing Bucket RotorsFixed Angle RotorsControl SystemDrive SystemRotor Locking SystemImbalance Detection SystemProgramsPulse (Short) RunAcceleration /Deceleration RatesCentrifugation ChamberMax Timer RangeTemperature RangePre-Cooling FunctionRefrigeration SystemSound LevelOther Features1 Biocontainment certiﬁcation by the Public Health England, Porton Down, UKFits in. Stands out.Fits in.More centrifuge solutions for your labPerfect Fit. It’s simple. From 1 Liter bottles, to 15 and50 mL conical tubes and microplates, the versatile selection ofThermo Scientiﬁc™ Nalgene™ and Nunc™ centrifugationproducts work seamlessly with your complete centrifuge androtor system, bringing together outstanding quality and performance.Thermo Scientiﬁc Heraeus Benchtop CentrifugesAccelerate your research with high speeds, rotor versatility and outstanding safety. Fromintuitive controls, to the certiﬁed“click” in place, Thermo Scientiﬁc Heraeus benchtop centrifuges deliver the perfect blendof capabilities to support micro-volume protocols, a wide range of high volume swing-outapplications and high speed processing.Thermo ScientiﬁcSelect from two available speed options, up to 1721,000 x g for high-end research applications, and our extensive rotor portfolio, including:•1 Biocontainment certiﬁcation by the Public Health England, Porton Down, UK。

Amicon Ultra-15 Centrifugal Filter Devices

English IntroductionAmicon® Ultra-15 centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors and easy concentrate recovery from dilute and complex sample matrices. Thevertical design and available membrane surface area provide fast sample processing, high sample recovery (typically greater than 90% of dilute starting solution), and the capability for 80-fold concentration.Typical processing time is 15 to 60 minutes depending on Nominal Molecular Weight Limit (NMWL). Solute polarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the filter device prevents spinning to dryness and potential sample loss. The concentrate is collected from the filter device sample reservoir using a pipettor, while the ultrafiltrate is collected in the provided centrifuge tube. The device can be spun in a swinging bucket or fixed angle rotor. Amicon® Ultra-15 devices are supplied non-sterile and are for single use only.The Amicon® Ultra-15 product line includes 5 different cutoffs (Nominal Molecular Weight Limit, NMWL, or Molecular Weight Cutoff, MWCO):●Amicon® Ultra 3K device — 3,000 NMWL ●Amicon® Ultra 10K device — 10,000 NMWL ●Amicon® Ultra 30K device — 30,000 NMWL ●Amicon® Ultra 50K device — 50,000 NMWL ●Amicon® Ultra 100K device — 100,000 NMWLAmicon® Ultra-15 10K filter devices are for in vitro diagnostic use and can be used to concentrate serum, urine, cerebrospinal fluid, and other body fluids prior to analysis.Amicon® Ultra-15 3K, 30K, 50K, and 100K filter devices are for research use only and not for use in diagnostic procedures.User GuideAmicon ® Ultra-15Centrifugal Filter Devicesfor volumes up to 15 mLAmicon® Ultra-15 10K device for in vitro diagnostic use Amicon® Ultra-15 3K, 30K, 50K, and 100K devices forresearch use only; not for use in diagnostic proceduresMaterials SuppliedThe Amicon® Ultra-15 device is supplied with a cap, a filter device, and a centrifuge tube.Applications●Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA samples, either single- or double-stranded), microorganisms, column eluates, and purified samples ●Purification of macromolecular components found in tissue culture extracts and cell lysates, removal of primer, linkers, or molecular labels from a reaction mix, and protein removal prior to HPLC ●Desalting, buffer exchange, or diafiltrationRequired Equipment●Centrifuge with swinging bucket or fixed angle rotor with wells/carriers that can accommodate50 mL tubes CAUTION : To avoid damage to the device during centrifugation, check clearance before spinning. ●Pipettor with 200 microliter (μL) tip for concentrate recoverySuitabilityPreliminary recovery and retention studies are suggested to ensure suitability for intended use. See the “How to Quantify Recoveries” section.Device Storage and Shelf LifeStore at 15–30 °C. Shelf life is 3 years from date of manufacture. For cat. nos. UFC901008, UFC901024, and UFC901096, refer to expiration date on package label.PrerinsingThe ultrafiltration membranes in Amicon® Ultra-15 devices contain trace amounts of glycerine. If this material interferes with analysis, prerinse the device with buffer or Milli-Q® water. If interference continues, rinse with 0.1 N NaOH followed by a second spin of buffer or Milli-Q® water. CAUTION: Do not allow the membrane in Amicon® Ultra filter devices to dry out once wet. If you are not using the device immediately after prerinsing, leave fluid on the membrane until the device isused.How to Use Amicon® Ultra-15 Centrifugal Filter Devices1. Add up to 15 mL of sample (12 mL if using a fixed angle rotor) to the Amicon® Ultra filter device.2. Place capped filter device into centrifuge rotor; counterbalance with a similar device.3. When using a swinging bucket rotor, spin the device at 4,000 × g maximum for approximately15–60 minutes.When using a fixed angle rotor, orient the device with the membrane panel facing up and spin at 5,000 × g maximum for approximately 15–60 minutes.NOTE: Refer to Figures 1 and 2, and Tables 1 and 2 for typical spin times.4. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdrawthe sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation. Desalting or DiafiltrationDesalting, buffer exchange, or diafiltration are important methods for removing salts or solvents in solutions containing biomolecules. The removal of salts or the exchange of buffers can be accomplished in the Amicon® Ultra-15 device by concentrating the sample, then reconstituting the concentrate to the original sample volume with any d esired solvent. The process of “washing out” can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced. See example below.1 mg/mL100 mMNaCl200 μL of75 mg/mLprotein inNaCl200 μL of75 mg/mLprotein inNaClAdd 14.8 mL of10 mM NaClor exchangebufferSpin conditions: 4,000 × g, room temperature, 15 mL starting volume.Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6.Spin conditions: 5,000 × g, room temperature, 12 mL starting volume.Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6.PerformanceFlow RateFactors affecting flow rate include sample concentration, starting vol u me, chemical nature of solute, relative centrifugal force, centrifuge rotor angle, membrane type, and temperature. Figures 1 and 2, and Tables 1 and 2 can be used to estimate the time required to achieve a given volume of filtrate orconcentrate for a variety of protein markers. A typical spin time for a 15 mL sample is approximately 15 to 60 minutes (depending on device nominal molecular weight limit). While most of the sample is filtered in the first 15 to 30 minutes of centrifugation, the lowest concentrate volume (150–300 μL) is reached after spinning for 15 to 60 minutes.Flowrate, continuedTable 1. Typical Concentrate Volume vs. Spin Time (Swinging bucket rotor)Spin conditions: 4,000 × g, room temperature, 15 mL starting volume. Protein markers used: Cytochrome cfor 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6 (mean value of 3 membrane lots). Shaded volumes were used for the calculation of protein recovery in Table 4.Table 2. Typical Concentrate Volume vs. Spin Time (Fixed angle rotor)Spin conditions: 5,000 × g, room temperature, 12 mL starting volume. Protein markers used: Cytochrome cfor 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6 (mean value of 3 membrane lots). Shaded volumes were used for the calculation of protein recovery in Table 4.Protein Retention and Concentrate RecoveryThe membranes used in Amicon® Ultra devices are characterized by a nominal molecular weight limit (NMWL); that is, their ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the NMWL may be only partially retained. Membrane retention depends on the solute’s molecular size and shape. For most applications, molecular weight is a convenient parameter to use in assessing retention characteristics. Merck Millipore Ltd. recommends using a membrane with a NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. Refer to Table 3.Table 3. Typical Retention of Protein Markers Spin Conditions for Tables 3 and 4: Swinging bucket rotor (4,000 × g, 15 mL starting volume), or fixed angle rotor,(5,000 × g, 12 mL starting volume), room temperature, n=6 (mean value of 3 membrane lots).Protein Retention and Concentrate Recovery, continuedFactors that determine sample recovery include the nature of the protein solute relative to the device NMWL chosen, starting concentration, and concentration factor. Table 4 provides typical recoveries forAmicon® Ultra-15 devices.Table 4. Typical Concentrate Recovery Concentrate Concentration Concentrate Shaded volumes were taken from Tables 1 and 2.How to Quantify RecoveriesCalculate total recovery, percent concentrate recovery, and percent filtrate recovery using the method below. The procedure provides a close approximation of recoveries for solutions having concentrations up to roughly 20 mg/mL.NOTE : Appropriate assay techniques include absorption spectrophotometry, radioimmunoassay, refractiveindex, and conductivity.Direct Weighing ProcedureThe density of most dilute proteins is nearly equal to the density of water (i.e., 1 g/mL). Using this property, the concentrate and filtrate volumes can be quantified by weighing them and converting the units from grams to milliliters. This technique is valid only for solutions with concentrations of approximately 20 mg/mL or less.1. Before use, separately weigh the empty filter device, the centrifuge tube, and an empty tube forconcentrate collection.2. Fill filter device with solution and reweigh.3. Assemble device and centrifuge per instructions.4. Collect the concentrate with a pipettor and dispense it into the preweighed concentrate collectiontube.5. Remove the device from the centrifuge tube and weigh the centrifuge tube and concentrate collectiontube.6. Subtract weight of empty device/tubes to calculate weights of starting material, filtrate, andconcentrate.7. Assay the starting material, filtrate, and concentrate to determine solute concentration.8. Calculate recoveries using the weight/volume data and the measured concentrations as follows:Maximizing Sample RecoveryLow sample recovery in the concentrate may be due to adsorptive losses, over-concentration, or passage of sample through the membrane.●Adsorptive losses depend upon solute concentration, its hydrophobic nature, temperature and timeof contact with filter device surfaces, sample composition, and pH. To minimize losses, remove concentrated samples immediately after centrifugal spin.●If starting sample concentration is high, monitor the centrifugation process in order to avoid over-concentration of the sample. Over-concentration can lead to precipitation and potential sample loss. ●If the sample appears to be passing through the membrane, choose a lower NMWL Amicon® Ultra-15device.Direct Weighing Procedure, continued% concentrate recovery = 100 × % filtrate recovery = 100 × % total recovery = % concentrate recovery + % filtrate recovery W c = total weight of concentrate before assay W o = weight of original starting material W f = weight of filtrate C c = concentrate concentrationC o = original starting material concentration C f = filtrate concentrationW c × C c W o × C oW f × C fW o × C oSpecificationsMaximum initial sample volumeSwinging bucket 15.0 mL Fixed angle rotor 12.0 mL Typical final concentrate volume 150–300 μL Maximum relative centrifugal forceSwinging bucket rotor 4,000 × g Fixed angle rotor 5,000 × g Active membrane area 7.6 cm 2DimensionsFilter device in tube (capped)Length: 122 mm (4.8 in.)Diameter: 29.7 mm (1.2 in.)Filter deviceLength: 72.0 mm (2.8 in.)Diameter: 29.6 mm (1.2 in.)Materials of ConstructionFilter device Copolymer styrene/butadiene Membrane Ultracel® low binding regenerated cellulose Filtrate tube Polypropylene Filtrate cap and liner PolyethyleneChemical CompatibilityAmicon® Ultra centrifugal devices are intended for use with biological fluids and aqueous solutions. Before use, check the sample for chemical compatibility with the device.Table 5. Chemical Compatibility of Amicon® Ultra Filter DevicesAcids Concentration ConcentrationAcetic acid ≤ 50%*Phosphoric acid ≤ 30%Formic acid ≤ 5%*Sulfamic acid≤ 3% Hydrochloric acid ≤ 1.0 M Sulfuric acid ≤ 3%Lactic acid ≤ 50%Trichloroacetic acid (TCA)≤ 10%*Nitric acid ≤ 10%Trifluoroacetic acid (TFA)≤ 30%*AlkalisAmmonium hydroxide ≤ 10%Sodium hydroxide ≤ 0.5 M Alcoholsn-Butanol ≤ 70%Isopropanol ≤ 70%Ethanol ≤ 70%Methanol ≤ 60% DetergentsAlconox® detergent ≤ 1%Sodium dodecyl sulfate (SDS)≤ 0.1% CHAPS detergent≤ 0.1%Tergazyme®detergent ≤ 1%Lubrol® PX detergent≤ 0.1%Triton® X-100 surfactant≤ 0.1% Nonidet™ P-40 surfactant≤ 2%Tween® 20 surfactant≤ 0.1%Sodium deoxycholate≤ 5%Organic solventsAcetone not recommended Ethyl acetate not recommended Acetonitrile ≤ 20%Formaldehyde ≤ 5% Benzene not recommended Pyridine not recommended Carbon tetrachloride not recommended Tetrahydrofuran not recommended Chloroform not recommended Toluene not recommended Dimethyl sulfoxide (DMSO)≤ 5%*MiscellaneousAmmonium sulfate Saturated Phenol≤ 1%Diethyl pyrocarbonate ≤ 0.2%Phosphate buffer (pH 8.2)≤ 1 M Dithiothreitol (DTT)≤ 0.1 M Polyethylene glycol≤ 10% Glycerine≤ 70%Sodium carbonate ≤ 20% Guanidine HCl ≤ 6 M Tris buffer (pH 8.2)≤ 1 M Imidazole≤ 100 mM Urea ≤ 8 M Mercaptoethanol≤ 0.1 M* Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are recommended to determine whether leachables represent potential assay interferences.In Vitro Diagnostic Product LabelingThe following table defines the symbols found on Amicon® Ultra-15 10K device labels.Product Ordering InformationThis section lists the catalogue numbers for Amicon® Ultra Ultrafiltration Devices. See the Technical Assistance section for contact information. You can purchase these products on-line at/products.* Amicon® Ultra-4 and -15 10K devices are for in vitrodiagnostic use. All other devices are for research use only.11Amicon® Ultra-15 Centrifugal Filter Devices NoticeThe information in this document is subject to change without notice and should not be construed as a commitment by Merck Millipore Ltd. (“Millipore”) or an affiliate. Neither Merck Millipore Ltd. nor any of its affiliates assumes responsibility for any errors that may appear in this document.Technical AssistanceFor more information, contact the office nearest you. Up-to-date world-wide contact information is available on our web site at /offices. You can also visit the tech service page on our web site at /techservice.Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms (within the “Terms and Conditions of Sale” applicable to your purchase transaction).The M logo is a trademark of Merck KGaA, Darmstadt, Germany. Millipore, Amicon, Milli-Q, and Ultracel are registered trademarks of Merck KGaA. Alconox and Tergazyme are registered trademarks of Alconox, Inc. Lubrol is a registered trademark of Imperial Chemical Industries PLC. Nonidet is a trademark of Royal Dutch/Shell Group. Triton is a registered trademark of Union Carbide Corp. Tween is a registered trademark of ICI Americas Inc.© 2012 EMD Millipore Corporation. All rights reserved.PR03520TR, Rev. A, English, 03/12Made in IrelandMerck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, IRL。

Amicon Pro Affinity Concentration Kit - GST 产品说明书

Amicon® Pro AffinityConcentration Kit - GSTPurification of GST-tagged recombinant proteins.Catalog Nos. ACR5000GS, ACK5003GS, ACK5010GS, ACK5030GS,ACK5050GS, ACK5100GSFOR RESEARCH USE ONLYNot for use in diagnostic procedures.USA & Canada Phone: +1(800) 437-7500 Fax: +1 (951) 676-9209Australia +61 3 9839 2000IntroductionThe expression and purification of recombinant proteins is central to protein regulation, structure and function studies. Purification has been greatly facilitated by the addition of small affinity tags to protein sequences such as the glutathione S-transferase domain (GST). GST agarose resin is an affinity chromatography matrix, when used in concert with the Amicon®Pro Affinity Concentration device, offers rapid purification of GST fusion proteins, native GST, or glutathione-binding proteins. GST-tagged proteins bind specifically to reduced glutathione in near-neutral, non-denaturing conditions (e.g., phosphate buffer). Following resin capture of the target protein, unbound lysate components are removed by spin-based clearing and washing steps. Bound protein is recovered by centrifugation following competitive displacement with buffer containing free, reduced glutathione.By condensing the entire purification workflow into one device, the Amicon® Pro device eliminates the need for multiple sample transfers thereby minimizing protein loss. The large exchange device reservoir (up to 9ml) accommodates a range of sample capacities as well as reduces the need for multiple spin steps during the wash and elution phases. Direct coupling to an Amicon® Ultra-0.5 device further provides simultaneous concentration during the elution phase. Lastly, the Amicon® Pro device offers highly efficient diafiltration in a single 15 minute spin.The kit contains sufficient GST resin, optimized buffers, and Amicon® Pro devices for 12 standard reactions.Sample Preparation GuidelinesOptimizing lysis parameters is critical for protein extraction and downstream purification. The Amicon®Pro Affinity Concentration GST kit is compatible with a range of conditions, including reducing agents (β-mercaptoethanol, DTT), chelating agents (EDTA), non-ionic detergents, and BugBuster®Protein Extraction Reagent (Cat. No. 70584), a proprietary mixture of non-ionic detergents offering a rapid cost-effective alternative to mechanical cell lysis methods such as sonication.Irrespective of extraction method, we recommend inclusion of rLysozyme™ Solution (Cat. No.71110) and Benzonase®Nuclease (Cat. No. 70746) and during protein extraction for increased cell lysis (protein yield) and a reduction in sample viscosity (improved sample handling), respectively.Protease inhibitors may also be added to the lysis buffers to protect against degradative enzymes.A listing of individual protease inhibitors and cocktails is available at /search on key words “Protease Inhibitor.” Note: Serine proteases should be used with caution if the GST fusion tag is to be cleaved via Thrombin or Factor Xa digestion. If used during protein extraction, we strongly recommend the eluted fraction be buffer exchanged prior to performing the cleavage reaction.In certain systems, high protein expression can lead to aggregation in the form of inclusion bodies.Strong denaturants such as 6 M guanidine or 8 M urea can be used to solubilize aggregates greatly enhancing yield. However, only properly folded, functional GST is capable of binding GST resin. GST fractions recovered from denaturation of inclusion bodies must be refolded to reconstitute active GST fusion constructs. To a degree, this can be accomplished through use of the Protein Refolding Kit (Cat. No. 70123).Cat. No. BWEGSFor more information, consult the GST•Bind™ Resin User Guide available at /chemicals. Search using the Cat. No. 70541.Kit ComponentsCS211421-GST Resin (3 mL) – Crosslinked agarose with immobilized reduced glutathione supplied as 50% slurry in 50 mM Phosphate Buffer pH 7.5, 0.15 M NaCl, 0.1% NaN3. The resin utilizes an 11-atom spacer arm (~16 Å) for covalent attachment of reduced glutathione via a sulfide linkage. The binding capacity is typically 5-8 mg/mL of settled resin.Buffers:o CS211416-10X GST Bind/Wash Buffer (25 mL) – 43 mM Na2HPO4, 14.7 mM KH2PO4,1.37 M NaCl, 27 mM KCl pH7.2o CS211354-10X Glutathione Reconstitution buffer (5 mL) – 500 mM Tris, pH 8.0o CS211418-Glutathione, reduced, free acid (125 mg)Amicon®Pro Devices – The kit includes 12 complete assemblies. Each device consists of an exchange device, holder tube, 50 mL collection tube, and Amicon®Ultra-0.5 centrifugal filter device. A 2 mL collection tube is included for sample recovery from the AU-0.5 device by reverse spin. The kit is available in five formats based on the nominal molecular weight limit (NMWL) of the AU-0.5: 3 (ACK5003GS), 10 (ACK5010GS), 30 (ACK5030GS), 50 (ACK5050GS), and 100 kDa (ACK5100GS). Consult the Amicon®Pro (/psp, search keywords “Amicon Pro”) and Amicon®Ultra-0.5 centrifugal filter device User Guides (/catalogue/module/c82301) for proper assembly/disassembly and additional product information.All reagents should be stored at 2° to 8°C (do not freeze). The Amicon Pro devices can be stored separately at room temperature.Procedures for using the Amicon® Pro Affinity Concentration Kit - GSTThe protocol is based on purification of GST-tagged protein from 1 mL of E. coli lysate using 200 µl of GST resin slurry (100 µL packed resin). The protocol is linearly scalable for 50-1000 µL of resin slurry. Due to large variability among sample preps, parameters which may require optimization include bead input, binding time, wash, and elution parameters. This protocol includes steps for simultaneous concentration during the elution step as well as buffer exchange using the Amicon®Ultra-0.5 centrifugal filter device.Note: Given the collection tube’s capacity, it is not necessary to remove filtrate between the various centrifugation steps. However, if process samples need be retained for analytical purposes, the collection tube should be cleared.Buffer Preparation10X Bind/Wash Buffer should be diluted to 1X with sterile deionized H2O; 1X Bind/Wash solution may be stored at 4°C for 1 week.Prepare 10X GST Elution Buffer containing 100 mM reduced glutathione as follows: add 4ml 10X Reconstitution buffer directly to the glutathione powder vial. Pipette repeatedly then incubate for30 minutes at room temperature. Divide buffer into working volumes and store at -20o C (400Cat. No. BWEGSµl/standard reaction); 10X GST Elution Buffer is stable for 6 months at -20o C. We do not recommend repeated freeze/thaw cycles. To minimize glutathione oxidation, dilute 10X buffer to 1X using sterile deionized water and pH to 8.0 immediately before use. Discard any unused 1X Elution Buffer.Bead Preparation1. To ensure uniform suspension, vortex the GST resin thoroughly before adding it to the device.2. Remove the collection tube cap and open the exchange device cap.3. Add 200 µL of resin slurry to the base of the exchange device. Close the exchange cap.Up to 500 µl packed resin (1000 µl slurry volume) may be added per device We recommend using wide-bore tips (Cat. No. 02-707-134, Fisher Scientific) for resin transfer.4. To remove storage buffer, centrifuge in a swinging bucket rotor at 1000 g X 1 min.5. Add 500 µL of 1X Bind Buffer. Centrifuge at 1000 g X 1 min.Protein Binding1. Add 500 µL of sample to the exchange device.Up to 9 mL of sample can be added. The volume loaded is determined by the target protein’s expression level and resin’s binding capacity.2. Incubate for 60 min at room temp with gentle agitation.We recommend upright agitation on a plate shaker at low setting.End-over-end mixing, particularly with small volumes or for extended time, may result in substantial bead loss to the sides of the feeder tube.The duration of binding time may vary with application.3. Centrifuge the device at 1000 g X 1 min in a swinging bucket rotor. Recover the sample flow-through from the 50 mL collection tube (optional).To ensure maximal protein capture, collect all resin into solution prior to centrifugation.4. Add1.5 mL of Wash Buffer. Centrifuge at 1000 g X 1 min. Recover the wash fraction from the 50mL collection tube (optional).Due to the large capacity of the exchange device, the volume of the wash can be increased for greater sample purity. There is no need for multiple wash steps.Sample ElutionSamples can be eluted without concentration by adding elution buffer and centrifuging (1000g X 2 minute) directly into a clean 50 ml collection tube. Given the limited volume processing capacity of the AU-0.5 device, we recommend this protocol if elution volumes > 1.5 ml are required.For simultaneous elution with concentration, attach the Amicon® Ultra-0.5 device and follow the steps outlined below.1. Remove the exchange device and insert it into the AU0.5 device.2. Place the exchange device/AU-0.5 assembly back in the holder and return the device to thecollection tube.3. Add up to 1.5 mL of Elution Buffer, gently resuspend the resin, and incubate for 5 min.Under standard conditions, one elution is sufficient for recovery of 90-95% of captured protein.4. Close the exchange device cap and screw on the collection tube cap to ensure a proper seal.5. Centrifuge at 4000 g X 15 min in a swinging bucket rotor. Concentrated samples can be bufferexchanged or recovered from the AU-0.5 device by reverse spin (see below).Cat. No. BWEGSDepending on the starting elution volume, NMWL of AU0.5 device employed, and the degree of concentration desired, the length of the spin time can range for 10-30 minutes. Please consult the Performance Characteristics section in the Amicon®Pro Affinity Concentration System User Guide (/psp, and search keywords "Amicon Pro”) for recommended guidelines., consult.6. Recover the concentrated fraction by reverse spin or proceed to Buffer Exchange (see below).Buffer Exchange (Optional if samples have been collected in the Amicon® Ultra-0.5 device)1. After sample concentration, add 1.5ml desired buffer to the exchange device/AU-0.5 assembly.2. Centrifuge device at 4000g X 15 minutes in a swinging bucket rotor. Concentrated samples canbe recovered from the AU-0.5 device by reverse spin (see below).Collect sample from the AU0.5 device by Reverse Spin(following Concentration or Buffer Exchange)1. Disassemble the exchange device/AU-0.5 assembly from the holder tube.2. Using a gentle twisting motion, detach the AU-0.5 from the exchange device.3. If there is residual sample in the exchange device tip, depress the exchange device cap to expelthe remaining sample volume into the AU-0.5.4. Hold AU-0.5 upright and slide the 2 ml collection tube on top of it.5. Invert the assembly and centrifuge (in a microcentrifuge) with a fixed angle rotor 1000g X 2min.Sample protein yield can be determined by Mid IR-based spectrometry using the DirectDetect™ biomolecular quantitation system and DirectDetect™ Assay Free Sample Cards.TroubleshootingCat. No. BWEGSIssue: Recombinant Protein is present in low amount in eluatePossible Cause SolutionProtein is insoluble and may have formed inclusion bodies. After lysate clearance, check both the supernatant and pellet for protein. Perform lysis and binding procedures under denaturing conditions.The GST fusion protein is not in the proper three dimensional conformation. Attempt to reconstitute native protein structure using Protein Refolding Kit (Cat. No. 70532).The GST-tag is not exposed for binding to the affinityresin.The protein may require denaturing conditions for binding.The GST-tag is not present. Sequence the ligation junctions to ensure that the readingframe is correct. Check for possible internal translation starts(N-terminal tag) or premature termination sites (C-terminal tag). Recombinant protein is degraded during cell lysis. Add protease inhibitors during cell lysis.Protein forms aggregates. Add solubilizing agents such as detergents (0.1% Triton® X-100, TWEEN®-20) or increase salt concentration.pH of the Lysis or Binding buffers is incorrect. Check buffer pH; the acceptable range is 7-8. Acidic bufferswill prevent binding.Protein expression is insufficient. Optimize the growth/induction conditions.Cell Lysis is incomplete. Optimize the Cell Lysis Protocol.Cell Lysate is too viscous. If possible, dilute the lysate in Bind Buffer. Alternatively,include Benzonase® Nuclease during lysis to remove freeRNA/DNA.Protein precipitates during sample concentration while using the AU0.5 device due to over-concentration. Reduce the duration of the centrifugation time during the elution/concentration step.Protein is lost during sample concentration while using AU0.5 device. Check the protein's expected size and MWCO of AU0.5 device used. AU0.5 device is offered in 5 different MWCO formats - 3, 10, 30, 50, and 100 kDa.Issue: High Non-specific bindingPossible Cause SolutionInsufficient washing. Increase the volume of the Wash Buffer used or the number ofwash steps. Alternatively, supplement the Bind/Wash Buffers.Contaminants interact directly with the GST fusion protein. Add reducing agents such as DTT or β-mercaptoethanol to reduce disulfide bonds. Add detergents to disrupt non-specific interactions.GST fusion protein is degraded. Degraded/truncated forms of the recombinant protein will stillbind to the GST resin and appear as contaminating bands inSDS-PAGE. Perform a lysis procedure on ice and includeprotease inhibitors.Cell lysate is too concentrated. Dilute the lysate in Bind Buffer before purification.Cat. No. BWEGSCat. No. BWEGSPerformanceProduct Ordering InformationNo Devices Amicon ® Pro + AU 0.5 ml with MWCO: 3k 10k 30k 50k 100k Amicon ® Pro Affinity Concentration Kit - GSTACR5000GS ACK5003GS ACK5010GS ACK5030GS ACK5050GS ACK5100GS Amicon ® Pro AffinityConcentration System 12PKACS500312 ACS501012 ACS503012 ACS505012 ACS510012 Amicon ® Pro AffinityConcentration System 24PK ACS500324 ACS501024 ACS503024 ACS505024 ACS510024The Amicon ® Pro Affinity Concentration Kit contains reagents and devices sufficient for 12 standard reactions. Amicon ® Pro devices are also sold separately in 12 and 24 packs.Description Catalogue Number Qty/Pack GST•Bind™ Resin 70541-3/4/5 10/50/25 mL GST•Bind™ Buffer Kit 70534 1 KitPurification using Amicon Pro Affinity Concentration Kit – GST purifications of a 50 kDa GST-tagged protein the Amicon ® Pro Affinity Concentration Kit numbered lanes are: 2 – flowthrough fraction 3 – wash fraction 4 – eluted fractionNoticeThe information in this document is subject to change without notice and should not be construed as a commitment by EMD Millipore Corporation (“Millipore”) or an affiliate. Neither EMD Millipore nor any of its affiliates assumes responsibility for any errors that may appear in this document. Technical AssistanceFor more information, contact the office nearest you. Up-to-date world-wide contact information is available on our web site at /offices. You can also visit the tech service page on our web site at /techserves.WarrantyEMD Millipore Corporation(“EMD Millipore”) warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products. EMD MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications and descriptions of EMD Millipore products appearing in EMD Millipore’s published catalogues and product literature may not be altered except by express written agreement signed by an officer of EMD Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon.In the event of a breach of the foregoing warranty, EMD Millipore Corporation’s sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies EMD Millipore Corporation promptly of any such breach. If after exercising reasonable efforts, EMD Millipore Corporation is unable to repair or replace the product or part, then EDM Millipore shall refund to the Company all monies paid for such applicable Product. EMD MILLIPORE CORPORATION SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS.Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.(c) 2009 - 2012: Merck KGaA, Darmstadt. All rights reserved. No part of these works may be reproduced in any form without permission in writing.Benzonase®, BugBuster®, and Amicon® are registered trademarks of Merck KGaA, Darmstadt, Germany. DirectDetect™ and GST•Bind™ is a trademark of Merck KGaA, Darmstadt, Germany. Triton® is a registered trademark of Dow Chemical Company. Tween® is a registered trademark of ICI Americas, Inc.Cat. No. BWEGS。

PureProteome Protein A和Protein G磁性珠粒体数据表说明书

PureProteome Protein A and Protein G magnetic beads are a powerful system toisolate antibodies faster, easier, and with more reproducibility then ever before. PureProteome Protein A and Protein G magnetic beads purify your sample with the highest binding capacity of any magnetic beads available. You can achieve reproducible results in both immunoprecipitation and serum depletion assays.PureProteome ™ Protein A and Protein G Magnetic BeadsEliminate Variability. Maximize Recovery.AdvantagesHigh capacity: More than 6x the • binding capacity of competitive magnetic beads.Bind 1.5-3.5 µg of rabbit IgG –per µL of suspensionConsistent results: Total removal • of buffers with no sample loss Fast processing time: Bead • immobilization occurs in seconds Ideal for immunoprecipitation and • serum depletion assaysEconomical: Up to half the price of • competitive magnetic beads2Rabbit Serum (50 µL) diluted with PBS was incubated withProtein G magnetic beads per manufacturer’s instructions. Thedepleted rabbit serum samples were separated by SDS-PAGEand the gel stained with Coomassie Blue using Millipore andcompetitor magnetic beads. Lane 1: input material, lanes 2, 4, 6,8: depleted samples, lanes 3, 5, 7, 9: eluted samples.A whole-cell lysate (300 mg of protein in 500 µL) preparedfrom A431 cells was incubated in the presence of 5 µg of amouse monoclonal antibody specific for the tumor suppressorprotein/transcription factor p53 for 2 hours at 4 °C withgentle mixing. The antigen-antibody complexes were thenincubated for 10 minutes at room temperature with 50 µL ofpre-washed PureProteome Protein A magnetic beads followedby washes with PBS to remove any unbound protein. Theimmunoprecipitated p53 protein was eluted from the beadsby incubation in the presence of gel loading buffer for 10minutes at 70 °C. Ten microliter samples of the cell lysatestarting material (S), cell lysate that had been incubated in thepresence of the PureProteome magnetic beads (FT) and thep53-containing eluted fraction (E) were subjected to SDS-PAGE and western blot analysis using Immobilon-P blottingmembrane. The immunoreactive p53 protein was visualizedusing the SNAP i.d.™ Protein Detection System (antibodies:rabbit anti-p53, HRP-conjugated goat anti-rabbit) and ImmobilonHRP Western Substrate. The results shown demonstratequantitative precipitation of the p53 protein from the samples. HigH BinDing CApACitywitH RApiD SAMplE pRoCESSingTrust Millipore to develop a superior magnetic bead systemoptimized to advance your research. Millipore’s process forparamagnetizing porous silica particles enables us to providemagnetic beads with the highest binding capacity whencompared to other magnetic purification systems.With a binding capacity 6x greater then competitive beads,PureProteome Protein A and Protein G magnetic beadsprovide the greatest percentage of immunoglobulin (IgG)depletion from serum samples with low non-specific binding.get up to 8X greater depletion in half the time. Superior magnetic bead performancefrom the experts in porous media3CoMplEtE RECoVERy witH REpRoDuCiBlE RESultSMillipore’s new PureProteome magnetic beads ensure the rapid and reproducible isolation of proteins. Unlike conventional methods that require centrifugation to pellet followed by careful aspiration to avoid sample loss, PureProteome magnetic beads are isolated using a magnetic rack. This allows for the total removal of buffers for complete recovery of beads with no sample dilution.Eliminate variabilitywhile maximizing recovery.FASt AnD EASyWhile traditional methods require minutes of harsh centrifuga-tion to isolate your sample, the PureProteome magnetic bead system isolates proteins in seconds using a magnetic rack right on your bench. A magnetic field gently immobilizes the highly-visible beads on the side of the tube. This allows quick and easy aspiration and eliminates the need for a centrifugation step. With the increased reaction kinetics of the Pure Proteome magnetic beads, incubations can be performed in minutes.Immunoprecipitation reactions that may require up to 18 hours of incubation with beads using conventional methods can now be accomplished in as little as 10 minutes.Dramatically reduce your sample preparation time with pureproteome magnetic beads.oRDERing inFoRMAtionDescription Qty/pk Catalogue no. PureProteome Magnetic BeadsPureProteome Protein A Magnetic Beads10 mL2 x 1 mLLSKMAGA10LSKMAGA02PureProteome Protein G Magnetic Beads10 mL2 x 1 mLLSKMAGG10LSKMAGG02Pure Proteome Nickel Magnetic Beads10 mL2 x 1 mLLSKMAGH10LSKMAGH02Magna GrIP™ Rack20-400Description nMwl*Qty/pk Catalogue no. Protein Concentration (< 4 mL samples)Amicon® Ultra-4Centrifugal Filter Unit withUltracel®-3 membrane324UFC800324Amicon Ultra-4 CentrifugalFilter Unit with Ultracel-10membrane1024UFC801024Amicon Ultra-4 CentrifugalFilter Unit with Ultracel-30membrane3024UFC803024Amicon Ultra-4 CentrifugalFilter Unit with Ultracel-50membrane5024UFC805024Amicon Ultra-4 CentrifugalFilter Unit with Ultracel-100membrane10024UFC810024 * Nominal Molecular Weight Limit or membrane cut-off in kDaDescription Qty/pk Catalogue no. Western BlottingSNAP i.d.™ Protein Detection SystemIncludes hardware base, blot holdersample pack, blot roller, vacuum tubinguser guide.1 kit WBAVDBASEDescription Size Qty/pk Catalogue no. Immobilon®-P Transfer membrane (0.45 µm)Cut Sheet8 x 10 cm10IPVH0810010 x 10 cm10IPVH1010020 x 20 cm10IPVH20200 Roll26.5 x 375 cm1IPVH00010 Immobilon-PSQ Transfer membrane (0.2 µm)Cut Sheet8 x 10 cm10ISEQ0810010 x 10 cm10ISEQ1010020 x 20 cm10ISEQ20200 Roll26.5 x 375 cm 1 ISEQ00010 Immobilon-FL Transfer membrane (0.45 µm)Cut Sheet10 x 10 cm 10 IPFL10100 Roll26.5 x 3.75 cm 1 IPFL00010 Blotting SandwichesImmobilon-PBlotting Sandwich7 x 8.4 cm 20 IPSN078528.5 x 13 cm 20 IPSN08132 Description Qty/pk Catalogue no. Western Blot Detection SubstratesImmobilon WesternChemiluminescent HRP Substrate100 mL WBKLS0100Immobilon WesternChemiluminescent AP Substrate100 mL WBKDS0100Spray & Glow™ ECL Western BlottingDetection System100 mL17-373 Visit for additional pack sizes/offices to plACE An oRDER oR RECEiVE tECHniCAl ASSiStAnCEVisit: /supportMillipore, Ultracel, Immobilon, and Amicon are registered trademarks of Millipore Corporation. The M mark, Advancing Life Science Together, PureProteome, Magna GrIP, SNAP i.d., and Spray & Glow are trademarks of Millipore Corporation.Lit. No. DS1060EN00 11/08 BP-GEN-08-01172Printed in the U.S.A.© 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.。

msd sulfo-tag nhs-ester-说明书

MSD GOLD™ SULFO-TAG NHS-EsterMSD GOLD SULFO-TAG™ NHS-Ester 150 nmol (sufficient for conjugating 1 mg of IgG) R91AO-12 µmol (sufficient for conjugating 10 mg of IgG) R91AO-2MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs Pack 1 (sufficient for conjugating 5 x 200 µg of IgG) R31AA-1 Pack 2 (sufficient for conjugating 5 x 1 mg of IgG) R31AA-2MSD Labeling ReagentsMSD GOLD SULFO-TAG NHS-EsterFor labeling aminesNote:150 nmol size of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugating 1 mg of IgG and the 2 µmol size is sufficient for conjugating 10 mg of IgG at a challenge ratio of 20.Each MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack contains enough material for 5 reactions. At a challenge ratio of 20, Pack 1 is sufficient for conjugating 200 µg of IgG per reaction, and Pack 2 is sufficient for conjugating 1 mg of IgG per reaction.This package insert must be read in its entirety before using this product.FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.MESO SCALE DISCOVERY®A division of Meso Scale Diagnostics, LLC.1601 Research BoulevardRockville, MD 20850-3173 USAMESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, U-PLEX, V-PLEX, S-PLEX, STREPTAVIDIN GOLD, MESO, , SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), U-PLEX (design), V-PLEX (design), S-PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC. ©2015 Me s o Scale Diagnostics, LLC. All rights reservedTable of ContentsIntroduction (4)Preparation of MSD GOLD SULFO-TAG Conjugates (5)MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs (6)Additional Materials and Equipment (6)Protocol (8)Storage, Handling, and Stability (10)FAQs (11)Worksheet (14)Ordering InformationMSD Customer ServicePhone: 1-240-314-2795Fax: 1-301-990-2776Email: CustomerService@MSD Scientific SupportPhone: 1-240-314-2798Fax: 1-240-632-2219 attn: Scientific SupportEmail: ScientificSupport@IntroductionThis protocol details the conjugation procedure for proteins of molecular weight (MW) > 40,000 D altons using MSD GOLD SULFO-TAG NHS-Ester label. The straightforward procedure involves an optional buffer exchange step, a 2-hour incubation step, and a mandatory buffer exchange step to quickly isolate the conjugated protein using a spin column. MSD GOLD SULFO-TAG NHS-Ester (Figure 1) is an amine reactive, N-hydroxysuccinimide ester which readily couples to primary amine groups of proteins under mildly basic conditions to form a stable amide bond.MSD GOLD SULFO-TAG conjugates are stable and may be used at low concentrations. These features minimize time, cost, and labor as large batches of a stable conjugate can be prepared, validated, and used for long periods of time. Its excellent performance characteristics and simple conjugation procedure make MSD GOLD SULFO-TAG NHS-Ester the product of choice for molecules that contain primary amines (e.g., lysine-containing proteins). MSD GOLD SULFO-TAG offers low non-specific binding, resulting in highly sensitive detection when used in conjunction with MSD instruments.Figure 1: MSD GOLD SULFO-TAG NHS-EsterPreparation of MSD GOLD SULFO-TAG ConjugatesGeneral NotesIn order to minimize hydrolysis of MSD GOLD SULFO-TAG NHS-Ester, the reagent should be dissolved in cold distilled water just prior to its addition to the protein solution. If necessary, the stock MSD GOLD SULFO-TAG NHS-Ester solution can be kept on ice for up to 10 minutes. The reconstituted solution is unstable and any unused material should be discarded. Consider conjugating more than one protein at the same time to maximize the use of the MSD GOLD SULFO-TAG NHS-Ester reagent. Generally, 150 nmol of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugation of up to 1 mg of IgG.The labeling ratio is the number of molecules of MSD GOLD SULFO-TAG conjugated to each molecule of protein. Optimal conjugation ratios for a MSD GOLD SULFO-TAG conjugated protein should be determined empirically for each specific application. For most applications using IgG antibodies (MW ~150,000), optimal performance is obtained with conjugation ratios between 2:1 and 20:1. In this range, assay signals typically show a linear dependence on conjugation ratio. Conjugation ratios significantly higher than 10:1 can be counterproductive and may lead to elevated background signals or loss of binding activity. For proteins that are significantly smaller than IgGs, lower conjugation ratios (between 1:1 and 5:1) may provide better assay performance.The challenge ratio is the number of moles of MSD GOLD SULFO-TAG per mole of protein in the conjugation reaction mixture. The challenge ratio required to achieve a specific conjugation ratio depends on a number of factors including pH, temperature, protein concentration, protein size, and the number of lysines available for coupling. Conjugating a 2 mg/mL IgG solution using the standard conditions described in this protocol will typically result in a label incorporation of approximately 50% (i.e., a challenge ratio of 10:1 will result in a conjugation ratio of about 5:1). Conjugation efficiencies for other proteins may be different. In general, conjugating with high protein concentrations of 1–2 mg/mL in slightly alkaline PBS (pH 7.9) in the absence of preservatives yields the best conjugation efficiencies. Maintaining consistent conjugation conditions (protein concentration, buffer type, MSD GOLD SULFO-TAG NHS-Ester concentration, incubation time, and temperature) is important when preparing multiple batches of conjugated protein in order to achieve consistent assay results.When developing immunoassays, MSD recommends conjugating antibodies using the standard conditions outlined in this document and challenge ratios of 6:1, 12:1, and 20:1 to identify optimal conjugation conditions. If evaluating different conditions is not possible due to limited reagent quantities, a challenge ratio of 20:1 will generally provide good performance. For immunogenicity applications where an antibody drug or protein therapeutic is used, the suggested challenge ratios are 12:1 and 6:1 MSD GOLD SULFO-TAG:drug. If only one ratio is tested, a 10:1 challenge ratio is recommended. For details on building immunogenicity assays, please refer to the Bridging Immunogenicity Guidelines for Assay Development at . The protocol describes the MSD GOLD SULFO-TAG conjugation procedure for proteins with a MW > 40,000 D a. Smaller proteins/polypeptides may also be conjugated using MSD GOLD SULFO-TAG NHS-Ester as long as they have an accessible lysine or the N-terminal amino group; however, alternative separation methods may be needed to remove unconjugated label. MSD offers a variety of services for the custom conjugation of reagents including proteins, peptides, and non-proteinaceous molecules.MSD GOLD SULFO-TAG NHS-Ester Conjugation PacksMSD offers all-inclusive conjugation packs that include the components and guidance that may be necessary for conjugating and purifying detection reagents with MSD GOLD SULFO-TAG label. Two sizes of conjugation packs are offered: MSD GOLD SULFO-TAG Conjugation Pack 1 and Pack 2. The two packs enable the conjugation of different amounts of IgG. MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 1 contains materials for conjugation and purification of up to 200 µg of IgG per reaction, and MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 2 allows conjugation of up to 1 mg of IgG per reaction. Each pack contains enough material for 5 reactions (i.e., 5 vials of MSD GOLD SULFO-TAG NHS-Ester).Table 1. Components of MSD GOLD SULFO-TAG Conjugation Packs*MSD GOLD SULFO-TAG Conjugation Pack 1 includes 10 columns of 0.5 mL capacity and 10 syringes of 1 mL size, and MSD GOLD SULFO-TAG Conjugation Pack 2 includes 10 columns of 5 mL capacity and 10 syringes of 3 mL size.**The pH of Conjugation Buffer is stable for up to 2 weeks after opening the bottle. For long term use, it is recommended to readjust the pH of the solution.Additional Materials and EquipmentThe following additional materials may be required. Some items (denoted with a *) are included in the MSD GOLD SULFO-TAG Conjugation Packs.1.Conjugation Buffer* (Phosphate-buffered saline (PBS), pH 7.9, preservative-free)2.Conjugate Storage Buffer* (PBS pH 7.4 + 0.05% Sodium Azide)3.Polypropylene microfuge tubes4.Spin columns.* MSD recommends the use of Zeba Spin Desalting Columns, 40K MWCO of various sizes from ThermoScientific, Catalog # 87766–877735.15 mL conical tubes for use with Zeba Spin Desalting Columns, 40K MWCO, 2 mL column size6.Protein assay such as BCA, Bradford, or Lowry7.MSD Blocker A (optional), Catalog # R93AA-2 (250 mL) and R93AA-1 (1 L)8.Spectrophotometer capable of an OD455 measurementReagent Storage Size Quantity DescriptionMSD GOLD SULFO-TAG NHS-Ester ≤-70°C 150 nmol 5 vials MSD GOLD SULFO-TAG NHS-Ester label forcoupling to antibodies and other proteinsZeba Spin Desalting Column, 40K MWCO* 2–8°C 0.5 mL or5 mL10 columns Size exclusion chromatography columns for thepurification of proteins larger than 40,000 DaFilter, 0.22 µm RT N/A 10 each Filter for use during purification Syringe * RT N/A 10 each Syringe for use during purificationConjugation Buffer** 2–8°C 40 mL 1 bottle Phosphate-buffered saline (PBS), pH 7.9, preservative-freeConjugate Storage Buffer RT 40 mL 1 bottle PBS pH 7.4 + 0.05% Sodium Azide9. 0.2 µm filter* (optional) Table 2. Suggestions for 0.2 µm filter10. Concentrator (optional) Table 3. Suggestions for concentratorsNoteThe following table lists the catalog numbers of the Zeba Spin Desalting Columns, 40K MWCO, from Thermo Scientific and the recommended sample volume for each column.Table 4. Catalog numbers of Zeba Spin Desalting Columns from Thermo ScientificVendorCatalog # Volume of Conjugated ProteinWhatmanAV125EAQU ≥ 2.0 mL Millipore or Fisher Scientific (MILLEX-GV)SLGV004SL0.2–1.0 mLVendorCatalog # Range Millipore BIOMAX-50 concentrator, 50 MWCO UFV5BQK25 0.05–0.5 mL AMICON Ultra-4 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC805008 0.5–4.0 mL AMICON Ultra-15 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC9050240.5–15.0 mLThermo ScientificCatalog #Number of Columns/packZEBA Spin Desalting Column VolumeRecommended Volume of the Conjugation Reaction87766 25 0.5 mL 70–100 µL 87767 50 0.5 mL 70–100 µL 87768 5 2 mL 200–450 µL 87769 25 2 mL 200–450 µL 87770 5 5 mL 300–1,000 µL 87771 25 5 mL 300–1,000 µL 87772 5 10 mL 1,000–2,000 µL 877732510 mL1,000–2,000 µLProtocol1.Prepare a 1–2 mg/mL solution of the protein to be conjugated in Conjugation Buffer. Antibodies in a storage buffer withpreservatives such as sodium azide or EDTA must be buffer exchanged before the conjugation reaction. It is recommended that dilute protein solutions be concentrated to at least 1 mg/mL. Protein solutions should be concentrated and/or buffer exchanged using the spin columns described above or an alternative centrifugal filtration/concentration unit that has been equilibrated with preservative-free PBS, pH 7.9. (Use Conjugation Buffer when using MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack.) Filter the protein using a 0.2 µm filter. The concentration of the protein solution to be conjugated should be confirmed prior to beginning the conjugation reaction.Note: Conjugation buffer, 0.2 µm filter, and syringes are included in the MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs.2.Equilibrate the protein to be conjugated with MSD GOLD SULFO-TAG NHS-Ester at the conjugation temperature of 23°C.A temperature range of 20°C to 25°C is acceptable. The equilibration can take between 10-30 minutes depending on thevolume of protein.3.Calculate the amount of MSD GOLD SULFO-TAG NHS-Ester stock solution required for the conjugation reaction using theformula depicted below and on the attached worksheet.EXAMPLE❑500 µL of 2 mg/mL antibody❑12:1 challenge ratio❑MSD GOLD SULFO-TAG stock = 3 nmol/µL1000 x 2 mg/mL x 12 x 500 µL = 80 nmol of MSD GOLD SULFO-TAG reagent required150,000 Da80 nmol of SULFO-TAG reagent = 26.7 µL of SULFO-TAG stock solution required for conjugation reaction3 nmol/µL SULFO-TAG stock solution4.Centrifuge the MSD GOLD SULFO-TAG NHS-Ester vial by pulse spinning for 1 minute or gently tap on a soft surface inorder to collect lyophilized material at the bottom of the vial. Reconstitute MSD GOLD SULFO-TAG NHS-Ester immediately prior to use with cold distilled water. For the 2 µmol and 150 nmol sizes of MSD GOLD SULFO-TAG NHS-Ester, dissolve with 200 µL and 50 µL, respectively to generate stock solutions of 10 and 3 nmol/µL. Gently vortex the vial to ensure complete dissolution of all lyophilized material.Note: Reconstituted MSD GOLD SULFO-TAG NHS-Ester may be kept for up to 10 minutes on ice prior to use.5.Add the calculated volume (from Step 3) of reconstituted MSD GOLD SULFO-TAG NHS-Ester to the protein solution andvortex immediately. Discard any remaining unused MSD GOLD SULFO-TAG NHS-Ester.6.Incubate at 23°C for 2 hours; a temperature range of 20°C to 25°C is acceptable. Shield the reaction from light bycovering the tube with aluminum foil or placing it in a dark area (e.g., a closed drawer). Take care to maintain consistent conjugation conditions between multiple preparation lots to ensure conjugation reproducibility.7.Prepare Zeba Spin D esalting Columns, 40K MWCO, towards the end of the incubation period. Remove the column'sbottom closure and loosen the cap. Do not remove the cap. Place the column in a collection tube to remove the storage buffer and wash the column 3 times with Conjugate Storage Buffer. Each preparation step should be carried out by centrifuging the columns, and their respective collection tubes, in a centrifuge with a swinging bucket rotor at 2–8°C.Refer to Table 5 below for the recommended sample volume, wash buffer volumes, collection tube sizes, and centrifugation times for each preparation step.Note: Reaction volumes larger than the capacity of a Zeba column should be distributed over multiple Zeba columns.8.Apply the conjugation reaction to the Zeba column in a dropwise manner following the recommendations in Table 2. Usinga swinging bucket rotor, centrifuge the columns in clean new collection tubes to purify the MSD GOLD SULFO-TAGconjugated protein. The MSD GOLD SULFO-TAG conjugated protein will be captured in the conical tubes. Retain the conjugated material in the conical tubes, and discard the columns.Note: The unconjugated MSD GOLD SULFO-TAG reagent will appear as yellow in the spin column.Table 5: Specifications for Zeba Spin Desalting Columns, 40K MWCOSize of Column (mL) 0.5 2 5 10 Sample Volume Range (µL) 70–100 200–450 300–1,000 1,000–2,000 Wash Buffer Volume 300 µL 1 mL 2.5 mL 5 mL Sample Volumes to use a stacker1N/A <350 µL <750 µL <1,500 µL Optional Stacker Volume N/A 40 µL 100 µL 200 µL Centrifugation Speed 1,500 x g1,000 x g1,000 x g1,000 x gCentrifugation Time(Min) Storage Solution Removal 1 2 2 2 Wash 1 1 4–8 4–8 4–8 Wash 2 1 2–8 2–8 2–8 Wash 32 3 5–8 5–8 5–8 Sample Recovery33–4 6–8 6–8 6–8addition of the protein to the column.2 If column is not mostly white after the third wash, the column may be spun for an additional 1–3 minutes.3 Additional sample recovery spin time is allowed if needed to ensure maximum recovery of sample. Overdrying the resin at this stage will not harm the protein; therefore, spinning for up to 8 minutes is allowed.9.It is recommended to filter the conjugated protein using a 0.2 µm filter. Filtration may cause some loss of the protein.Please refer to page 7 for the recommended filter units.10.Determine the molar protein concentration of the conjugated protein using a standard colorimetric protein assay such asBCA, Bradford, or Lowry.absorbance reading as MSD GOLD SULFO-TAG will absorb light at this wavelength.Note: Do not use an OD28011.Measure the absorbance of the MSD GOLD SULFO-TAG protein conjugate at 455 nm using a spectrophotometer. Dividethe measured value by the path length in cm, and then divide by the extinction coefficient of the label (15,400 M-1cm-1) to obtain the MSD GOLD SULFO-TAG label concentration in moles per liter. For reference, a formula calculation worksheet page is attached.12.To calculate the MSD GOLD SULFO-TAG label:protein conjugation ratio, divide the MSD GOLD SULFO-TAG labelconcentration value determined in step 11 by the molar protein concentration value determined in step 10.13.Antibody conjugates are usually stable for at least 2 years at 2–8°C at a concentration of 1–2 mg/mL; stability of otherprotein types should be determined. Conjugated proteins may be stored frozen at ≤-20°C or ≤-70°C, as long as the protein is stable to freeze-thaw cycles or stored in single-use aliquots. MSD GOLD SULFO-TAG conjugated proteins may be sensitive to extended exposure to light and should be stored in the dark or in amber or opaque vials. Short-term exposure of conjugates to light when carrying out assays is not a concern. If the protein concentration is low (< 0.1 mg/mL), consider adding a carrier protein such as 0.1% MSD Blocker A.Storage, Handling, and StabilityMSD GOLD SULFO-TAG NHS-Ester is supplied as a dry orange-red lyophilized solid. The vials should be stored at ≤-70°C. The expiration date of the product is indicated on the label. Following reconstitution, any remaining unused material should be discarded.FAQs1)What chemicals interfere with MSD GOLD SULFO-TAG conjugation?Primary amines and strong nucleophiles interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation. Common reagents that can interfere with the amine coupling of NHS chemistry are:a)Trisb)Glycinec)Histidined)Azidee)Imidazolef)Glutathioneg)Ammonium ionsh)Glycerol2)What are typical carrier proteins in antibody solutions?a)BSAb)GelatinAntibodies should be obtained in carrier protein-free formulations for labeling with MSD GOLD SULFO-TAG NHS-Ester.Carrier proteins will interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation and cannot be removed with desalting columns.3)What is the minimum amount of material that can be conjugated?Generally, 50-100 µg can be conjugated in PBS (without interfering buffer components) if the protein concentration is high enough (1–2 mg/mL). Otherwise, microconcentrators may be used to concentrate the antibody solution following equilibration of the microconcentrator with PBS.4)Are there alternatives to using Thermo Scientific Zeba Spin D esalting Columns for purifying the MSD GOLDSULFO-TAG conjugated antibody after conjugation?a)Users may purchase commercially available G-50 SEPHADEX columns or prepare G-50 SEPHADEX columns atthe bench. However, some G-50 columns may not be efficient in complete removal of unconjugated material. TheSEPHADEX grade is important. MSD recommends using fine grade SEPHADEX for preparing self-packed gelfiltration columns. Medium Grade SEPHADEX does not provide suitable separations and Superfine SEPHADEXdoes not allow an adequate flow rate without use of a pump. It is not recommended to use PD10 columns or G-25 SEPHADEX spin columns for purification of MSD GOLD SULFO-TAG-conjugated protein as these are notable to separate free MSD GOLD SULFO-TAG reagent from labeled conjugates.b)Alternatively, CENTRICON concentrators or similar microconcentrator products with adequate MWCO (forconcentrator information please refer to page 7) can be used to remove unbound label. Resuspend theconjugation mixture in a larger volume of PBS-0.05% azide, concentrate to a smaller volume, and then repeat theprocess as per the product instructions for desalting applications.c)Post-conjugation purification of proteins with MW < 40,000 Da will require alternative procedures (such as high-resolution size exclusion chromatography, HPLC, FPLC, etc.) because ZEBA Spin Desalting Columns or G-50columns will not provide adequate separation in this size range.5)What is the molecular weight of MSD GOLD SULFO-TAG?Unreacted MSD GOLD SULFO-TAG NHS-Ester has a molecular weight of 1,141 g/mol. After the conjugation reaction, each conjugated MSD GOLD SULFO-TAG adds 1,027 g/mol to the protein.6)What types of material can be conjugated?MSD GOLD SULFO-TAG NHS-Ester is reactive with primary amines. Proteins and large peptides are easily labeled. Fab fragments have also been conjugated successfully.MSD Conjugation Services may be used to conjugate small molecules and peptides. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.7)What conjugation ratio is recommended for an IgM?A challenge ratio range from 8:1 to 12:1 may be used for conjugating IgM antibodies. The molecular weight of an IgM is inthe order of 900,000 Da. IgMs can be unstable at higher pHs, therefore conjugation at pH 7.0 to 7.2 may be better than the standard labeling buffer of pH 7.9 used for IgG.8)Are there alternatives to using Conjugation Buffer for the conjugation reaction?For best results, it is recommended to use Conjugation Buffer. However, other buffers can be used for the conjugation reaction provided they are free of amine-containing molecules (i.e., no Tris- or glycine-containing buffers) and preservatives. Affinity-purified antibodies are commonly eluted with high molarity glycine solutions; therefore, it is important that they are properly desalted prior to conjugation. Using alternative conjugation buffers may result in lower incorporation efficiencies.9)What should I do if my application requires the conjugated protein to be in a different buffer?The desalting columns may be equilibrated in a buffer other than PBS if the end application requires storage of the conjugated protein in a non-PBS buffer.10)Will my antibody retain activity after labeling?MSD GOLD SULFO-TAG is a small hydrophilic molecule and generally does not affect the function of its conjugation partner, especially when labeling large proteins such as antibodies. With small molecule or peptide labeling, the addition of MSD GOLD SULFO-TAG may have an effect on binding affinities.11)What is the stability of MSD GOLD SULFO-TAG NHS-Ester?The shelf life of MSD GOLD SULFO-TAG NHS-Ester is 3 years at ≤-70°C. The reagent can be stored for up to 2 years at ≤-10°C with minimal loss of activity. Reagent stability is lower at room temperature or at 2–8°C. At room temperature, there may be a 1/3 to 1/2 loss of active material in a month. Once the reagent is reconstituted, it should be used as soon as possible since the NHS-ester hydrolyzes in water. After reconstitution, the solution may be kept up to 10 minutes on ice with minimal loss of activity.12)What if the protein to be conjugated does not have any primary amine groups?Alternative linking chemistry options are available from MSD which allow non-amine containing molecules to be successfully labeled. These include Thiol-Reactive linker (SULFO-TAG Iodoacetamide), Carboxyl (-COOH) Reactive linker (SULFO-TAG Amine) and Carbohydrate Reactive SULFO-TAG. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.13)I have an IgG purified antibody from my protein production group which has been eluted into PBS. Should Idesalt before conjugation?Yes. Tris-glycine is a major component of antibody elution buffers used in the purification procedure. On many occasions,a single desalting into PBS is insufficient. It is recommended to repeat the desalting step into PBS to remove any tracequantities of Tris-glycine that can hinder conjugation with MSD GOLD SULFO-TAG.14)How do I conjugate a high concentration protein solution with MSD GOLD SULFO-TAG?The conjugation reaction will be more efficient at high protein concentrations. It is recommended to use a lower challenge ratio for conjugation to compensate for the increased efficiency.15)How do I conjugate a low concentration protein solution with MSD GOLD SULFO-TAG?MSD recommends the protein concentration to be at least 0.5 mg/mL. If concentrating the protein solution is not feasible, conjugation can be done at a lower concentration, which may result in lower conjugation efficiency. Therefore, the conjugation reaction should be performed at a challenge ratio of 20:1 or higher.16)How do I conjugate small proteins with MSD GOLD SULFO-TAG?Proteins with MW < 40,000 Da can be conjugated by the same chemistry as antibodies; lower challenge ratios may be required for MSD GOLD SULFO-TAG conjugation of small proteins and peptides than for IgGs. The NHS-Ester will react with primary amines such as lysine residues and the N-terminus of proteins and peptides. If there is no primary amine available, a different chemistry will be necessary. Post-conjugation purification of small proteins will require alternative procedures (such as HPLC, FPLC, etc.) because small proteins are not resolved by G-50 columns.17)Why can't I use a spectrophotometer at 280 nm to determine conjugated protein concentration?MSD GOLD SULFO-TAG strongly absorbs at 280 nm and will interfere with any measurement of protein concentration at this wavelength.18)What is the stability of MSD GOLD SULFO-TAG conjugated proteins?MSD GOLD SULFO-TAG conjugated protein is generally as stable as the unconjugated protein if it is stored in the appropriate buffer, concentration, and storage temperature. The conjugated protein should be stored in the dark, either at 2–8°C or frozen in aliquots. Azide should be added for long term storage at 2–8°C to prevent any microbial growth. If the protein concentration is low, consider adding a carrier protein, such as 0.1% MSD Blocker A.19)My antibody did not conjugate very well. What are the possible reasons?The presence of preservatives, carrier protein or residual Tris-glycine or other interfering substances in the conjugation buffer (see FAQ 1 and 2) can reduce conjugation efficiency of the protein. Very low concentrations of the starting material (below 0.5 mg/mL) may also reduce conjugation ratios. It has also been observed that some IgGs label more efficiently than others.20)What components can be removed by buffer exchange or dialysis?Salt, azide, glycerol, buffering agent (e.g., Tris), carbohydrates (e.g., trehalose), and amino acids (e.g., histidine, glycine) can be successfully removed by buffer exchange method.21)Who should I contact if I have any questions on MSD GOLD SULFO-TAG conjugation?Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.WorksheetDate:MaterialsProtein to be conjugatedConcentration: Vendor:Catalog number: Lot number:Sample PreparationMethod: Buffer:Lot number: Date:Columns/Concentrators: Lot number:MSD GOLD SULFO-TAG NHS-Ester ReconstitutionSize: Lot number:Distilled water:Lot number: Date:Volume of water added to vial: Stock concentration (nmol/µL):Separation and CalculationsBuffer:Lot number: Date:Columns: Lot number:Protein assay kit:Type: Lot number:Pre-Conjugation Calculations1,000 x Protein conc. (mg/mL) x Challenge ratio x Volume of protein solution (µL) = nmol of SULFO-TAG reagent required Protein MW (Da)nmol of SULFO-TAG reagent required = µL of SULFO-TAG stock solution required for conjugation reaction Conc. of SULFO-TAG stock solution (nmol/µL)。

密理博超滤管

用户指南
Amicon® Ultra-4 10K 离心过滤器
用于体积不超过4 mL的样本 UFC801008 UFC801024 UFC801096
C 用于体外诊断
中文
引言
Amicon® Ultra-4 10K离心过滤器具有快速超过滤功能，能够达到较高的浓缩系数，易于从稀释液和复杂的样本组合进行浓缩液回收。其竖式设计以及可用的滤膜表面积能提供快速的样本处理和较高的样本回收率（通常大于90%稀薄的初始溶液），并能进行80倍浓缩。典型的处理时间为10至20分钟。竖式设计将溶质极化和之后造成的滤膜结垢降至最低，过滤装置中的物理止滤点防止过滤器旋转过度使样本干燥和造成样本损失。浓缩液用移液管从过滤器的样本槽中收集，而超过滤滤出液则收集到所提供的离心管中。该装置可在摆桶（可提供最佳性能）或定角转子中旋转。 Amicon® Ultra-4 10K装置未经消毒，只供一次使用。
长度： 124 mm
直径： 17.3 mm
过滤器
长度： 73.4 mm
直径： 17.2 mm
装置材料
过滤器
苯乙烯共聚物/丁二烯
滤膜
Ultracel®低粘合性再生纤维素膜
滤出液管
聚丙烯
滤出液盖子和衬里
聚乙烯
ห้องสมุดไป่ตู้
Amicon® Ultra-4 10K离心过滤器
7
化学相容性
Amicon® Ultra超离心装置适用于生物液体及水溶液。使用前，请检查样本与装置的化学相容性。
表3. 典型的浓缩液回收率
分子量标准/ 浓度
细胞色素 c (0.25 mg/mL)
装置 MWCO
10K
旋转时间（分钟）
15

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立张博;李守军;杨保收【摘要】为了建立猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)抗体检测的间接ELISA方法,本研究以纯化的原核表达的PEDV截短N蛋白作为包被抗原,建立了PEDV抗体检测的间接ELISA方法,将该方法命名为rnPED-ELISA.该抗原不与其他常见的7种猪病的阳性血清发生交叉反应,批内和批间重复性试验的变异系数均小于13％;rnPED-ELISA相对于血清中和试验(SN)试验的敏感性为93.33％,特异性为90.00％;rnPED-ELISA与TSZ全病毒抗体检测试剂盒的符合率达91.67％.采用rnPED-ELISA方法检测200份临床样品,PEDV抗体阳性检出率为69.5％.本试验建立的rnPED-ELISA方法具有良好的敏感性和特异性,可为免疫猪群抗体监测和猪流行性腹泻流行病学调查提供一种快速、简便的血清学诊断方法.【期刊名称】《中国畜牧兽医》【年(卷),期】2016(043)006【总页数】7页(P1446-1452)【关键词】猪流行性腹泻病毒;重组截短N蛋白;间接ELISA;诊断【作者】张博;李守军;杨保收【作者单位】天津农学院动物科学与动物医学学院,天津 300384;农业部生物兽药创制重点实验室,天津 300308;天津瑞普生物技术股份有限公司研究院,天津300308;天津农学院动物科学与动物医学学院,天津 300384;农业部生物兽药创制重点实验室,天津 300308;天津瑞普生物技术股份有限公司研究院,天津 300308;天津农学院动物科学与动物医学学院,天津 300384;农业部生物兽药创制重点实验室,天津 300308;天津瑞普生物技术股份有限公司研究院,天津 300308【正文语种】中文【中图分类】S852.65+9.6猪流行性腹泻(porcine epidemic diarrhea，PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)引起的，是一种破坏肠道的传染病[1]，是冠状病毒属Ⅰ群的成员。

Abbott Molecular UltraPlex 1-Step ToughMix ROX 说明书

UltraPlex ® 1-Step ToughMix ® ROX™ (4X)DescriptionUltraPlex 1-Step ToughMix ROX is a ready-to-use, 4X-concentrated master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan ® 5’-hydrolysis probes on real-time PCR systems that utilize ROX passive reference with 490 nM excitation. First-strand cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. It is ideal for highly sensitive quantification of RNA viruses or low abundance RNA targets in uni- or multiplexed RT-qPCR applications as well as high throughput gene-expression studies. The system has been optimized to deliver maximum RT-qPCR efficiency, sensitivity, and specificity in reduced reaction volumes and fast cycle times. UltraPlex 1-Step ToughMix ROX contains all required components for RT-qPCR except RNA template and probe. It is compatible with all dual-labeled probe chemistries.qScript ® XLT is an engineered M-MLV reverse transcriptase (RT) with reduced RNase H activity for improved activity and stability at higher temperatures. The use of higher temperatures (50 to 54°C) for the first-strand step of one-step RT-qPCR provides higher specificity for primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis. These beneficial properties of qScript XLT RT are further enhanced by a hot-start mechanism for the reverse transcription step. Minimizing off-target extension by the RT during reaction assembly provides highly reproducible low copy quantification as well as extended room temperature stability of fully assembled reactions for high throughput operations.UltraPlex 1-Step ToughMix ROX is highly resistant to PCR inhibitors. A key component of the ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This provides an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific PCR artifacts.Instrument CompatibilityDifferent real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. UltraPlex 1-Step ToughMix ROX provides seamless integration on the Applied Biosystems 7000, 7300, 7700, 7900HT StepOne™, or StepOnePlus™. Please visit our web site at to find an optimized kit for your instrument platform(s).ComponentsReagent DescriptionUltraPlex 1-Step ToughMix ROX (4X) 4X reaction buffer containing dATP, dCTP, dGTP, dTTP, magnesium, qScript XLT reversetranscriptase, RNase inhibitor protein, hot-start DNA polymerase , stabilizers, and ROX Reference DyeStorage and Stability Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.Guidelines for One-Step RT-qPCRThe design of highly specific primers and probes is a critical parameter for successful one-step RT-qPCR. The use of computer aided primerdesign programs is encouraged in order to minimize the potential for internal secondary structure and complementation at 3’-ends within each primer, the primer pair, and primer/probe combinations. Regions of strong RNA secondary structure should be avoided as this can interfere with primer hybridization and/or impede procession of the reverse transcriptase. For best results, amplicon size should be between 70 and 150 bp. Optimal results may require titration of primer concentration between 400 and 900 nM. A final concentration of 450 nM each primer and 100 to 150 nM probe is effective for most applications. The efficacy and efficiency of any primer/probe set should be validated under fast cycling and/or rapid ramp rate protocols before use in qPCR studies.Cat No. 95167-100 Size: 100 x 20 µL reactions (500 µL)Store at -25ºC to -15°C protected from lightGuidelines for One-Step RT-qPCR continued:▪If frozen, thaw UltraPlex 1-Step ToughMix ROX on ice. Thoroughly mix by vortexing, and then centrifuge to collect contents to the bottom of the tube. Retain on ice before use.▪First-strand synthesis can be carried out between 42°C and 60°C. Optimal results are generally obtained with a 10-minute incubation at 50°C.Longer incubation times for first-strand synthesis (up to 20 min) may be used. Incubation at temperatures over 54°C may result in delayed Cqs for assays that are optimal at 48 - 50°C.▪We recommend a minimum of 30s incubation at 95°C to inactivate the RT and activate the hot-start polymerase prior to PCR cycling. Longer activation times (2 – 10 minutes) will not adversely affect product performance and may reduce early cycle background noise experienced with some hydrolysis probe chemistries.▪The kit is compatible with both fast or standard qPCR cycling protocols. Annealing and or extension temperatures may need to be optimized for a given primer/probe design or fluorogenic probe chemistry. Use the suggested protocol as a starting point. Multiplexed RT-qPCR may benefit froma slightly longer extension time (60 to 90s). Use of a slower ramp rate (~2.5°C/s) between the denaturation step and annealing/extension step mayimprove performance for some assays.▪To maximize specificity, reactions should be assembled and retained on ice before transfer to the qPCR instrument.▪Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reaction cocktail with all required components except RNA template and mix thoroughly by vortexing. Then, dispense equal aliquots into each reaction tube. Add RNA to each reaction as the final step. Addition of sample as 2 to 5-µL volumes will improve assay precision.▪Suggested input quantities of template are: 1 pg to 100 ng total RNA; 10 fg to 10 ng poly A(+) RNA; 10 to 1x108 copies viral RNA.▪After sealing each reaction, mix contents by vortexing, and then centrifuge briefly to collect components at the bottom of the reaction tube. Reaction AssemblyComponent Volume for 20-μL rxn. Final ConcentrationUltraPlex 1-Step ToughMix ROX (4X) 5 µL 1XForward primer(s) variable 300 – 900 nMReverse primer(s) variable 300 – 900 nMProbe(s) variable 50-200 nMNuclease-free water variableRNA template 2 to 10 µL variableFinal Volume (μL) 20 µLNote: For smaller, or larger, reaction volumes scale all components proportionally.RT-qPCR Cycling ProtocolIncubate complete reaction mix in a real-time PCR detection system as follows:cDNA Synthesis 50°C, 10 minInitial denaturation 95°C, 3 minPCR cycling (30 - 45 cycles) 95°C, 3s to 10s60°C, 30s to 90s (data collection step)‡‡Note: The use of longer extension times (90s at 60°C), or a 3-step cycling protocol with an extension step of 60s at 72°C can help mitigate suppression of a low copy amplicon when co-amplified with a high copy target sequence.Quality ControlKit components are free of contaminating DNase and RNase. UltraPlex 1-Step ToughMix ROX is functionally tested in duplexed RT-qPCR. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (r2≥ 0.990) and a PCR efficiency ≥ 90% for the primary GOI with constant detection for the limiting exogenous positive control assay.Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1.The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio, LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2.Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3.This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4.Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5.The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Quantabio, LLC.may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915; Telephone number: 1-888-959-5165.Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, Germany.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.UltraPlex, ToughMix and qScript are registered trademarks of Quantabio, LLC. TaqMan is a registered trademark of Roche Molecular Systems, Inc.. ROX is a trademark of Life Technologies Corporation.。

15ml超滤离心管使用说明书

Amicon® Ultra-15
Centrifugal Filter Devices
for volumes up to 15 mL
User Guide
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Amicon Ultra-15 10K device for in vitro diagnostic use Amicon Ultra-15 3K, 30K, 50K, and 100K devices for research use only; not for use in diagnostic procedures
Notice
The information in this document is subject to change without notice and should not be construed as a commitment by Millipore Corporation or an affiliated corporation. Neither Millipore Corporation nor any of its affiliated corporations assumes responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Millipore Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual. © 2009 MILLIPORE CORPORATION. ALL RIGHTS RESERVED. PR02842 Rev. B, 10/09

Amicon Ultra-2 中心分离滤芯设备用户指南说明书

Amicon ® Ultra-2 Centrifugal Filter Devices1User GuideAmicon ®Ultra-2Centrifugal Filter Devicesfor volumes up to 2 mLFor research use only;not for use in diagnostic procedures.IntroductionAmicon ® Ultra-2 centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors and easy concentrate recovery from dilute and complex sample matrices. The vertical design and available membrane surface area provide fast sample processing, high sample recovery (typically greater than 90% of dilute starting solution), and the capability for 50-fold concentration. Typical processing time is 10 to 60 minutes depending on Molecular Weight Cut Off (MWCO). Solutepolarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the filter device prevents spinning to dryness and potential sample loss. Efficient recovery of the concentrated sample (retained species) is achieved by a convenient reverse spin step after collecting the filtrate. The device can be spun in a swinging bucket or fixed angle rotor . Amicon ® Ultra-2 devices are supplied non-sterile and are for single use only.The Amicon ® Ultra-2 product line includes 5 different cutoffs (Molecular Weight Cut Off, MWCO). These devices are for research use only and not for use in diagnostic procedures. •Amicon ® Ultra 3K device — 3,000 MWCO •Amicon ® Ultra 10K device — 10,000 MWCO •Amicon ® Ultra 30K device — 30,000 MWCO •Amicon ® Ultra 50K device — 50,000 MWCO•Amicon ® Ultra 100K device — 100,000 MWCOApplications•Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA samples, eithersingle- or double-stranded), microorganisms, column eluates, and purified samples•Purification of macromolecular components found in tissue culture extracts and cell lysates, removal of primer , linkers, ormolecular labels from a reaction mix, and protein removal prior to HPLC •Desalting, buffer exchange, or diafiltrationMaterials SuppliedFilter DeviceConcentrate collection tubeFiltrate collectiontubeThe Amicon ® Ultra-2 device is supplied with two tubes. During operation, one tube is used to collect filtrate; the other to cap the device during concentration and subsequently to recover the concentrated sample.Amicon ® Ultra-2 Centrifugal Filter Devices2Required EquipmentCentrifuge with swinging bucket or fixed angle rotor with wells/carriers that can accommodate 17 mm × 100 mm tubes (same well/carrier size as for Amicon ® Ultra-4 devices and the former Centricon ® device).CAUTION: T o avoid damage to the device during centrifugation, make sure it is properly assembled and seated at the bottom ofthe rotor . The rim of the concentrate collection tube should be inside the rotor well. Check clearance before spinning.SuitabilityPreliminary recovery and retention studies are suggested to ensure suitability for intended use. See the “How to Quantify Recoveries” section.Device StorageStore at room temperature.PrerinsingThe ultrafiltration membranes in Amicon ® Ultra-2 devices contain trace amounts of glycerine. If this material interferes withanalysis, pre-rinse the device with buffer or Milli-Q ® water . If interference continues, rinse with 0.1 N NaOH followed by a second spin of buffer or Milli-Q ® water .CAUTION: D o not allow the membrane in Amicon ® Ultra filter devices to dry out once wet. If you are not using the deviceimmediately after pre-rinsing, leave fluid on the membrane until the device is used.How to Use Amicon ® Ultra-2 Centrifugal Filter Devices1. Insert the Amicon ® Ultra-2 device into the filtrate collection tube, making sure that the device is fully seated in the tube.2. Add up to 2 mL of sample to the device and cover with concentrate collection tube. Push the tube firmly onto the device.WARNING: FAdd sample3. collection tube is completely inside the well. See figures below. Counterbalance with a similar device.Orient device correctly in rotor Seat device fully in well4. Spin for approximately 10–60 minutes depending on the MWCO of the device used:4,000 × g maximum when using a swinging bucket rotor 7,500 × g maximum when using a fixed angle rotorNOTE:W hen spinning viscous solutions such as undiluted serum or plasma, do not exceed 5,400 x g.Refer to Figures 1 and 2 and Tables 2 and 3 for typical spin times.5. Remove the assembled device from the centrifuge and separate the Amicon ® Ultra filter device from the filtrate collection tube.6. To recover the concentrated solute, invert the Amicon ® Ultra filter device and concentrate collection tube. Place in centrifugeand counterbalance with a similar device. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device to the tube.NOTE: For optimal recovery, perform the reverse spin immediately.Amicon ® Ultra-2 Centrifugal Filter Devices3Invert device and concentrate tubeSeparate device from filtrate tube FiltrateConcentrateDesalting or DiafiltrationDesalting, buffer exchange, or diafiltration are important methods for removing salts or solvents in solutions containingbiomolecules. The removal of salts or the exchange of buffers can be accomplished in the Amicon ® Ultra-2 device by concentrating the sample, discarding the filtrate, then reconstituting the concentrate to the original sample volume with any d esired solvent. The process of “washing out” can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced. See example below.1 mg/mL protein in 100 mM NaCl50 µL of 40 mg/mLprotein in 100 mM NaCl 100 mM NaCl50 µL of 40 mg/mL protein in 12.3 mM NaCl12.3 mM NaCl10 mM NaCl or exchange buffe r2 mL of 1 mg/mL protein in 12.3 mMNaClPerformance - DNA ConcentrationThe Amicon ® Ultra-2 30K device provides the best balance between PCR recovery and PCR primer removal for double-stranded DNA for base pairs ranging from 137 to 1159.Table 1. Typical Recovery of Nucleotides from the Amicon ® Ultra-2 30K DeviceSwinging Bucket Rotor35° Fixed Angle Rotor 4,000 × g for 40 min7,500 × g, for 15 minPCR Product (base pairs)PCR Primer (bases)PCR Recovery (%)PCR Primer Removal (%)Final Voume (µL)PCR Recovery (%)PCR Primer Removal (%)Final Volume (µL) 137 10 20 4883 87 8692 80 6144 43 4178 75 7893 86 6727 22 25 115910 20 4896 97 9798 93 8235 39 3795 93 9598 93 8226 26 27100 µL PCR diluted to 2,000 µL starting volume, n=6Amicon ® Ultra-2 Centrifugal Filter Devices4Performance - Protein ConcentrationFlow RateFactors affecting flow rate include sample concentration, starting vol u me, chemical nature of solute, relative centrifugal force, centrifuge rotor angle, membrane type, and temperature. Figures 1 and 2 and Tables 2 and 3 can be used to estimate the time required to achieve a given volume of filtrate or concentrate for a variety of protein markers. A typical spin time for a 2 mL sample in a fixed angle rotor is approximately 10 to 60 minutes (depending on device nominal molecular weight limit). While most of the sample is filtered in the first 10 to 20 minutes of centrifugation, the lowest concentrate volume (30–70 μL) is reached after spinning for 10 to 60 minutes.Spin conditions: Swinging bucket rotor , 4,000 × g, room temperature, 2 mL starting volume. Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.Figure 2. Typical Filtrate Volume vs. Spin Time for Amicon ® Ultra-2 Device, Fixed Angle RotorSpin conditions: 35° fixed angle rotor , 7,500 × g, room temperature, 2 mL starting volume.Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.Table 2. Typical Concentrate Volume / Concentration Factor vs. Spin Time, Swinging Bucket RotorProtein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.Shaded volumes were used for the calculation of protein recovery in Table 5.Table 3. Typical Concentrate Volume/Concentration Factor v. Spin Time, Fixed Angle RotorProtein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.Shaded volumes were used for the calculation of protein recovery in Table 5.Amicon® Ultra-2 Centrifugal Filter Devices5Protein Retention and Concentrate RecoveryThe membranes used in Amicon® Ultra devices are characterized by a molecular weight cut off (MWCO); that is, their ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the MWCO may be only partially retained. Membrane retention depends on the solute’s molecular size and shape. For most applications, molecular weight is a convenient parameter to use in assessing retention characteristics. We recommend using a membrane with a MWCO at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. Refer to Table 4.Table 4. Typical Retention of Protein Markersroom temperature, n=12.Factors that determine sample recovery include the nature of the protein solute relative to the device MWCO chosen, starting concentration, and concentration factor. Table 5 provides typical recoveries for Amicon® Ultra-2 devices.Table 5. Typical Concentrate Recoveryvolume, room temperature, n=8.Maximizing Sample RecoveryLow sample recovery in the concentrate may be due to adsorptive losses, over-concentration, or passage of sample throughthe membrane.• A dsorptive losses depend upon solute concentration, its hydrophobic nature, temperature and time of contact with filter device surfaces, sample composition, and pH. To minimize losses, remove concentrated samples immediately after centrifugal spin.• If starting sample concentration is high, monitor the centrifugation process in order to avoid overconcentration of the sample.Over-concentration can lead to precipitation and potential sample loss.• If the sample appears to be passing through the membrane, choose a lower MWCO Amicon® Ultra-2 device.Amicon® Ultra-2 Centrifugal Filter Devices6Amicon ® Ultra-2 Centrifugal Filter Devices7How to Quantify RecoveriesCalculate total recovery, percent concentrate recovery, and percent filtrate recovery using the method below. The procedure provides a close approximation of recoveries for solutions having concentrations up to roughly 20 mg/mL.NOTE : A ppropriate assay techniques include absorption spectrophotometry, radioimmunoassay, refractive index, andconductivity.Direct Weighing ProcedureThe density of most dilute proteins is nearly equal to the density of water (i.e., 1 g/mL). Using this property, the concentrate and filtrate volumes can be quantified by weighing them and converting the units from grams to milliliters. This technique is valid only for solutions with concentrations of approximately 20 mg/mL or less.1. Separately weigh the empty filter device, filtrate collection tube, and concentrate collection tube before use.2. Fill filter device with solution and reweigh.3. Assemble device in filtrate collection tube and centrifuge per instructions.4. Collect the concentrate by reverse spin into the pre-weighed concentrate collection tube.5. Remove the device from the concentrate collection tube and weigh the filtrate and concentrate collection tubes.6. Subtract weight of empty device/tubes to calculate weights of starting material, filtrate, and concentrate.7. Assay the starting material, filtrate, and concentrate to determine solute concentration.8. Calculate recoveries using the weight/volume data and the measured concentrations as follows:% concentrate recovery = 100 × W c x C c W o x C o % filtrate recovery = 100 × W f x C f W o x C o% total recovery = % concentrate recovery + % filtrate recovery W c = total weight of concentrate before assay W o = weight of original starting material W f = weight of filtrateC c = concentrate concentrationC o = original starting material concentration C f = filtrate concentrationSpecificationsMaximum initial sample volume 2.0 mLTypical final concentrate volume 30–70 µL depending on MWCOMaximum relative centrifugal forceSwinging bucket rotor4,000 × g for concentration spin,1,000 × g for recovery spinFixed angle rotor7,500 × g for concentration spin,1,000 × g for recovery spinNOTE: W hen spinning viscous solutions such asundiluted serum or plasma, do not exceed 5,400 x g.Active membrane area 1 cm2Hold-up volume< 5 μLDimensionsFilter device and tubeLength (concentration mode; device in filtrate tube): 119.7 mm (4.71 in.)Length (recovery spin; device upside down in concentrate tube): 95.3 mm (3.75 in.)Filter device Diameter: 15.9 mm (0.63 in.)Length: 70.7 mm (2.78 in.)Filtrate tube Diameter: 13.8 mm (0.54 in.)Length: 52.9 mm (2.08 in.)Concentrate tube Diameter: 13.7 mm (0.54 in.)Length: 34.5 mm (1.36 in.)Materials of ConstructionFilter device Copolymer styrene/butadieneMembrane Ultracel® low-binding regenerated celluloseCollection tubes PolypropyleneAmicon® Ultra-2 Centrifugal Filter Devices8Chemical CompatibilityAmicon® Ultra centrifugal devices are intended for use with biological fluids and aqueous solutions. Before use, check the sample for chemical compatibility with the device.Table 6. Chemical Compatibility of Amicon® Ultra Filter Devices.Acids Concentration ConcentrationAcetic acid ≤ 50%*Phosphoric acid ≤ 30%Formic acid ≤ 5%*Sulfamic acid≤ 3%Hydrochloric acid ≤ 1.0 M Sulfuric acid ≤ 3%Lactic acid ≤ 50%Trichloroacetic acid (TCA)≤ 10%*Nitric acid ≤ 10%Trifluoroacetic acid (TFA)≤ 30%*AlkalisAmmonium hydroxide ≤ 10%Sodium hydroxide ≤ 0.5 MAlcoholsn-Butanol ≤ 70%Isopropanol ≤ 70%Ethanol ≤ 70%Methanol ≤ 60%DetergentsAlconox® detergent ≤ 1%Sodium dodecyl sulfate (SDS)≤ 0.1%CHAPS detergent≤ 0.1%Tergazyme® detergent ≤ 1%Lubrol® PX detergent ≤ 0.1%Triton® X-100 surfactant≤ 0.1%Nonidet™ P-40 surfactant ≤ 2%Tween® 20 surfactant≤ 0.1%Sodium deoxycholate≤ 5%Organic solventsAcetone Not recommended Ethyl acetate Not recommendedAcetonitrile ≤ 20%Formaldehyde ≤ 5%Benzene Not recommended Pyridine Not recommendedCarbon tetrachloride Not recommended Tetrahydrofuran Not recommendedChloroform Not recommended Toluene Not recommendedDimethyl sulfoxide (DMSO)≤ 5%*MiscellaneousAmmonium sulfate Saturated Phenol≤ 1%Diethyl pyrocarbonate ≤ 0.2%Phosphate buffer (pH 8.2)≤ 1 MDithiothreitol (DTT)≤ 0.1 M Polyethylene glycol≤ 10%Glycerine≤ 70%Sodium carbonate ≤ 20%Guanidine HCl ≤ 6 M Tris buffer (pH 8.2)≤ 1 MImidazole≤ 100 mM Urea ≤ 8 MMercaptoethanol≤ 0.1 M* C ontact with this chemical may cause materials to leach out of the component parts. Solvent blanks are recommendedto determine whether leachables represent potential assay interferences.Amicon® Ultra-2 Centrifugal Filter Devices9Product Ordering InformationThis section lists the catalogue numbers for Amicon® Ultra Ultrafiltration Devices. See the Technical Assistance section for contact information. You can purchase these products on-line at /products.MWCO Qty/pk Amicon®Ultra-0.5deviceAmicon®Ultra-2deviceAmicon®Ultra-4deviceAmicon®Ultra-15device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micon Ultra-4 and -15 10K devices are for in vitro diagnostic (IVD) use. All other devices are for research use only.Amicon® Ultra-2 Centrifugal Filter Devices10NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.Contact InformationFor the location of the office nearest you, go to /offices.Technical AssistanceVisit the tech service page on our web site at /techservice.Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms (“Conditions of Sale”).The vibrant M, Millipore, Amicon, Milli-Q, and Ultracel are trademarks of Merck KGaA, Darmstadt, Germany, or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.©2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All rights reserved.PR05484, Rev. 02/19Amicon® Ultra-2 Centrifugal Filter Devices11。

蛋白浓缩方法

请教：蛋白浓缩方法请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！可以用：超虑；盐析；过离子柱等等，看你的试验条件定咯蛋白浓缩方法基本有：丙酮沉淀法；免疫沉淀法；三氯醋酸沉淀法；硫酸铵沉淀法；（低温）有机溶剂沉淀法；聚乙二醇沉淀法；超滤法；透析法；离子交换层析和冷冻干燥法……1，丙酮沉淀法；三氯醋酸沉淀法试验要求的仪器简单，但是常常导致蛋白质变性。

2，免疫沉淀法：得有特异性抗体！3，硫酸铵沉淀法：利用高浓度盐将蛋白质析出（盐析）选择硫酸按是因为：盐析有效性，pH范围广，溶解度高，溶液散热少，经济！4，（低温）有机溶剂沉淀法：强调低温（0-4度以下）是因为10度时蛋白会在有机溶剂中变性，可用乙醇，丙酮……注意：Mg2+离子，pH值5，聚乙二醇沉淀法：使用PEG时旨在个别情况下才会是蛋白质稍有变性！他溶解是散热低，形成沉淀的平衡时间短，通常达到30%时蛋白质就会达到最大量的沉淀！6，超滤法：主要针对小体积蛋白质溶液（几ml）此法更不易引起变性！不过得有浓缩器，不是哪个实验室都有的！7，透析法：主要用于更换蛋白质的缓冲液！的有透析袋！不需要特殊的仪器！8，离子交换层析：可用阴离子交换树脂进行浓缩！9，冷冻干燥法：在冷冻状态下让扬品种的液体升华具体你的实验应该是做一种分泌蛋白吧？浓缩后要干什么，需要活性蛋白么？实验室设备如何，导师经费如何？这些都要考虑！！我用的是硫酸铵沉淀法，物廉价美。

用4度饱和硫酸铵等体积加入收集的上清中，以30000g的速度离心30分钟，弃上清，加入你所需体积的缓冲液体，在震荡器上使沉淀溶解，再在10000g下离心10分钟取上清分装保存于-80度即可关于蛋白浓缩，我认为最好的方法是真空冷冻干燥法，此法简便，蛋白质几乎无变性。

lhydy wrote:请教各位高手，我想将细胞培养上清中的蛋白浓缩10倍，但要确保最小的蛋白变性与蛋白丢失，应该采用什么方法比较好？请不吝赐教，谢谢！不知你何种用途，最简单的浓缩我们常用蔗糖或PEG包埋。

粪便-外泌体提取方法

centrifugalfilter units for 10 minutes at 4000x g.
Subsequently vesicles were stored in PBS at 80°C
feces were dissolved with PBS at 4◦C for 10 min,4 mL of PBS were used to dissolve every gram of feces,
15 min., 5000rpm, 4°C to remove solid debris,Two rounds
centrifugation steps from 500x g to 5,000x g at 10°C to remove debris.
the supernatant was filtered by the consecutive use of fourfilters with different pore sizes: 5.0, 1.2, 0.45mm0.2mm to remove the remaining cells/debris
harvested and diluted 30-fold with PBS.Exosome pellets were
resuspended in PBS and stored at -80˚C
supernatant was placed onto 1 mL of 0.8 M sucrose and 2.5 M sucrose in HEPES-buffered saline
centrifuged at 100,000×gfor 2 h at 4◦C, using an ultracentrifuge with SW32Ti rotor

lentivirus generation 慢病毒转染手册

Generation o f L entivirusReagents•293T: ATCC•Fugene 6 transfection reagent: Roche cat# 11814443001•FBS: Invitrogen cat# 16000-044•DMEM: Invitrogen cat# 11965-118•Pen/strep: Invitrogen cat# 15140-155•L-glutamine: Invitrogen cat# 25030-156•Non essential amino acid (NEAA): Invitrogen cat# 11140-050•Amicon Ultra Ultracel 100K: Millipore: cat# UFC910024•Beckman centrifuge tubes: Beckman: cat# 344058293T/fibroblast media (500 mls):DMEM (450 ml)10% FBS (50 ml)Pen/strep (5ml)L-glutamine (5ml)NEAA (5 ml)Reagent setupLentiviral vectors:Prepare all vector plasmids with Qiagen Maxiprep kits, including lentiviral packaging vectors and lentiviral transfer vectorsA. Production of Virus1. Seed 2x106 293T cells on a 100-cm tissue culture dish and incubate overnight until cellsreach ~70% confluence (~1-2 days). Replace with 10 ml of fresh media 2 hours before transfection.2. Mix lentiviral transfer vector and packaging vectors in 600 ul of DMEM in an eppendorftube. Add 50 ul of Fugene6 directly to the DNA mixture, making sure Fugene6 does not come in contact with the plastic. Gently vortex tube containing transfection mixture and incubate at RT for 20 min.Second generation packaging systemTransfer vector 10 ugpMD2.G 5 ugpsPAX2 5 ugThird generation packaging systemTransfer vector 10 ugpMDL g/pRRE 5 ugpRSV-Rev 2.5 ugpMD2.G 2.5 ug3. Add transfection mixture dropwise to cells; incubate 4 hrs to overnight (16 hours) andreplace with fresh medium.4. Collect virus-containing medium 48 hours after transfection and replace with freshmedium. Collect virus every 12 hours for up to 3 times total. Keep all viral media at 4Cuntil all collections are done. Pass viral media through a 0.45uM low protein-binding filter.At this point, the viral supernatant can be used to infect cells, frozen at -80C orconcentrated.Concentration using Amicon Ultra Centrifugal Filters:Transfer viral supernatnat to Amicon Filter and spin filter in tabletop centrifuge at 3000 rpm for 10-20 min at 4C. Concentrated virus can be aliquoted and stored at -80C. Thaw analiquot on ice before use; do not refreeze.Concentration by ultracentrifugation:1. Transfer viral supernatant to 33ml Beckman ultracentrifugation tubes and spin for 2hrs at 24,000 rpm using SW32Ti or SW28Ti.2. Pour off supernatant carefully and resuspend viral pellet using 100-ul pipette tip withappropriate amount of DMEM to concentrate virus 100x. It may take 30 min toovernight at 4C for the pellet to completely dissolve.3. Store virus in aliquots at -80C. Thaw an aliquot on ice before each use; do notrefreeze.。

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Contents
Introduction. .........................................................................................................................................................1 Applications..........................................................................................................................................................2 Materials Supplied................................................................................................................................................3 Required Equipment. ...........................................................................................................................................4 Suitability..............................................................................................................................................................4 Device Storage.....................................................................................................................................................4 Specifications.......................................................................................................................................................5 Chemical Compatibility.........................................................................................................................................7 How to Use Amicon Ultra-4 Centrifugal Filter Devices..........................................................................................9 How to Quantitate Recoveries............................................................................................................................10 Performance - DNA Concentration.....................................................................................................................13 Performance - Protein Concentration.................................................................................................................14 Flow Rate...................................................................................................................................................14 Protein Retention and Concentrate Recovery............................................................................................19 Maximizing Sample Recovery............................................................................................................................22 Desalting or Diafiltration.....................................................................................................................................23 Centrifugal Product Ordering Information...........................................................................................................24 Technical Assistance..........................................................................................................................................25 In Vitro Diagnostic Product Labeling...................................................................................................................26 Standard Warranty.............................................................................................................................................27
Introduction
Millipore’s Amicon® Ultra-4 centrifugal filter devices provide fast ultrafiltration, with the capability for high concentration factors and easy concentrate recovery from dilute and complex sample matrices. The vertical design and available membrane surface area provide fast sample processing, high sample recovery (typically greater than 90% of dilute starting solution), and the capability for 80-fold concentration. Typical processing time is 10 to 40 minutes depending on Nominal Molecular Weight Limit (NMWL). Solute polarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the filter device prevents spinning to dryness and potential sample loss. The concentrate is collected from the filter device sample reservoir using a pipettor, while the ultrafiltrate is collected in the provided centrifuge tube. The device can be spun in a swinging bucket (for optimal performance) or fixed angle rotor. Amicon Ultra-4 devices are supplied non-sterile and are for single use only.
Notice
The information in this document is subject to change without notice and should not be construed as a commitment by Millipore Corporation or an affiliated corporation. Neither Millipore Corporation nor any of its affiliated corporations assumes responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Millipore Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual. © 2009 MILLIPORE CORPORATION. ALL RIGHTS RESERVED. PR02843 Rev. B, 10/09