蛋白酶抑制剂Cocktail使用说明书奥默生物

合集下载

KOD酶使用说明书

用高成功率PCR酶「KOD FX」对发根样品的简便扩增例TOYOBO Co.,LTD 敦贺生物研究所杉山明生『KOD FX 』是在拥有各种优良特性的PCR 酶「KOD DNA polymerase 」的基础上开发的高性能PCR 酶。

本酶具有出色的「扩增成功率」、「扩增效率」和「延伸性」，可在各种扩增实验中得到确实有效的PCR 产物。

尤其是其出色的扩增成功率，在以粗样品为模板的PCR 中可得到充分的发挥。

比如，我们已通过实验确认，即便将全血、培养细胞等直接添加到PCR 反应液中，仍可得到良好的扩增。

扩增Mouse tail 、植物样品（叶块、米粒）时，只需将经简单前处理后配制的Lysate 添加到PCR 反应液中，即可获得确实有效的扩增产物。

也就是说，原来需事先从样品中纯化DNA 再进行PCR 实验的传统方法，如果使用『KOD FX 』，则无需进行DNA 纯化，只需简单的前处理即可进行PCR 扩增。

本篇中，作为粗样品和微量样品的实验例代表，我们尝试对发根样品进行简便的PCR 扩增。

由于发根样品可通过非侵袭性得到，因此不仅在法医学领域被应用，也在分子生物学实验中被应用。

但由于发根样品的量很少，因此DNA 含量也很少，而且毛发中的黑色素是抑制PCR 反应的物质，如进行DNA 纯化，则对技术和人力的要求非常高。

在这里，我们通过将『KOD FX 』和碱裂解法组合使用，不从发根样品中纯化DNA ，尝试进行简便的PCR 扩增。

以下为该实验的方法及结果。

（1）通过碱裂解法配制发根Lysate（2）PCR反应反应液组成发根 Lysate 人类循环方法将人的头发从发根处开始2mm 的地方切断，包含发根的部分作为样品。

用这样的样品5根，按图1所示的碱溶解法配制Lysate 。

请注意，用本方法不能使发根完全溶解（也无需完全溶解）。

另外，溶解液的核酸浓度无法测定。

取（1）配制的Lysate 2μl 直接添加到PCR 反应液中，按如下条件进行反应。

Western Blot基本原理、过程及注意事项之欧阳科创编

Western Blot基本原理、过程及注意事项原理是将蛋白质转移到膜上，然后利用抗体进行检测。

对已知表达蛋白，可用相应抗体作为一抗进行检测，对新基因的表达产物，可通过融合部分的抗体检测。

主要有三个过程：蛋白质电泳、转膜、检测。

样品的准备样品种类主要有培养的细胞、组织（需磨碎）、特殊细胞（切割下来的肿瘤细胞）等。

1、裂解液主要有RIPA和三去污两种，RIPA适用于细胞质中蛋白检测，而三去污适用于总蛋白。

三去污又分为离子型和非离子型。

离子型的裂解能力较强如SDS等，非离子型的包括NP-40、Tritonx-100。

2、裂解液的成分，3、样品缓冲液 sample buffer备注：1、样品应保持低温，且实验过程应低温操作，此举为了抑制酶的活性。

裂解完后样品液会显得粘稠是长结构DNA所致，故需要匀浆，打断DNA。

一般不用超声，因为容易引起蛋白不可逆的变性。

2、用2X样品缓冲液与样品1：1混匀。

4度保存。

3、样品混合液加样前需要100℃或沸水浴加热3-5分钟并迅速插入冰中，以充分变性蛋白。

配胶：胶的主要成分为丙烯酰胺和N，N’-亚甲双丙烯酰胺，应以温热(以利于溶解双丙稀酰胺)的去离子水配制含有29%(w/v)丙稀酰胺和1%(w/v)N，N’-亚甲双丙烯酰胺储存液丙稀酰胺29g，N，N-亚甲叉双丙稀酰胺1g，加H2O至100ml。

)储于棕色瓶，4℃避光保存。

4、分离胶的配方：以20ml的8﹪分离胶为例：5、浓缩胶配方：6ml为例备注：1、制胶是避免产生气泡，空气会影响胶的聚合，在蛋白质表观分子量分析时影响大。

2、因为有SDS，每次混匀时不应剧烈以免产生过多气泡。

3、蛋白容易与SDS脱离而失去负电荷,因此胶中加入SDS为了给蛋白持续的负电荷。

4、垫条清洗干净，与制胶板压紧，避免漏胶。

5、制胶时，加水应缓慢不要破坏胶面，其作用：可以平衡胶面，赶气泡等。

胶固定后用滤纸吸除干净，有水跑胶时条带会歪。

6、先插梳子再灌浓缩胶。

rLysozyme解毒液说明书

About the KitsrLysozyme Solution 300 KU 71110-31,200 KU 71110-4 6,000 KU 71110-5 rLysozyme Solution, Veggie™ Grade 1,200 KU 71297-46,000 KU 71297-5DescriptionrLysozyme Solution contains a highly purified and stabilized recombinant lysozyme that can beused for E. coli lysis. The enzyme catalyzes the hydrolysis of N-acetylmuramide linkages in thebacterial cell wall. The specific activity of rLysozyme Solution (1,700 KU/mg) is 250 times greater than that of chicken egg white lysozyme and therefore less enzyme is required to achieve E. colilysis. In contrast to chicken egg white lysozyme, rLysozyme is optimally active at a physiologicalpH (6.0–8.0) that is also compatible with Novagen's extraction reagents. Very small amounts ofrLysozyme Solution (3–5 KU/gram cell paste) enhance the efficiency of protein extraction withBugBuster ®, BugBuster HT, and PopCulture ® Protein Extraction Reagents (1). In the absence ofprotein extraction reagents, direct lysis of E. coli can be achieved by treatment of cell paste with 45–60 KU rLysozyme Solution in combination with a freeze/thaw treatment.rLysozyme Solution, Veggie Grade is a special grade of rLysozyme prepared using certifiedanimal-free or disease-free reagents. All of the steps in the preparation of the recombinantenzyme are carried out using reagents of non-animal origin, with the exception of the IPTG usedto induce protein expression. IPTG is chemically synthesized by a stringent process from D-galactose isolated from lactose, a milk sugar. The lactose is derived from certified disease-freecows. rLysozyme Solution, Veggie Grade has the same stability and specific activity as therLysozyme Solution and requires no changes to the protocol.This protocol describes E. coli lysis with rLysozyme treatment combined with a freeze/thawcycle of the cell pellet. Protocols for E. coli lysis using rLysozyme Solution with mechanical lysis,BugBuster ®, BugBuster HT, and PopCulture ® Protein Extraction Reagents are described inTechnical Bulletins 054 (His•Bind ® Kits), 245 (BugBuster Reagents), and 323 (PopCultureReagent). PopCulture extraction using rLysozyme and His•Tag ® or GST•Tag™ fusion proteinpurification in 96-well format can be achieved with RoboPop™ Purification Kits (Cat. Nos. 71102-3, 71103-3, 71188-3, 71189-3).Unit definition:One unit of rLysozyme is defined as the amount of enzyme that causes a decrease of 0.025 A 450 units per minute at 25°C in a 1.0 ml suspension (1 mg/ml) of Tuner™(DE3) cells in 0.5 ×BugBuster Reagent diluted with 50 mM Tris-HCl, pH 7.5. Note 1 KU = 1000 units.© 2003 Novagen ®, a brand of EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt, Germany. BugBuster, GST•Tag, His•Bind, His•Tag, Lysonase, PopCulture, rLysozyme, RoboPop, Tuner, Veggie, the Novagen logo and Novagen name are trademarks of EMD Biosciences, Inc. Benzonase is a trademark of Merck KGaA Darmstadt Germany. Triton is a trademark of Rohm and Haas Co.TB334 Rev. C 0503 United States & Canada 800-207-0144Germany ***********United Kingdom 0800 622935Or your local sales office Novagen Novagen products are sold for research use onlyComponents300 KU rLysozyme Solution• ••••• 300 KU rLysozyme Solution (30 KU/µl in 100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT,0.1 mm EDTA, 50% glycerol, 0.1% Tritron X-100, pH 7.5)1 ml rLysozyme Dilution Buffer (100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT,0.1 mM EDTA, 0.1% Triton X-100, pH 7.5)1,200 KU rLysozyme Solution1,200 KU rLysozyme Solution (30 KU/µl in 100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT,0.1 mm EDTA, 50% glycerol, 0.1% Tritron X-100, pH 7.5)6,000 KU rLysozyme Solution6,000 KU rLysozyme Solution (30 KU/µl in 100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT,0.1 mm EDTA, 50% glycerol, 0.1% Tritron X-100, pH 7.5)1,200 KU rLysozyme Solution, Veggie™ Grade1,200 KU rLysozyme Solution, Veggie Grade (30 KU/µl in 100 mM NaCl, 50 mM Tris-HCl,1 mM DTT, 0.1 mm EDTA, 50% glycerol, 0.1% Tritron X-100, pH 7.5)6,000 KU rLysozyme Solution, Veggie Grade6,000 KU rLysozyme Solution, Veggie Grade (30 KU/µl in 100 mM NaCl, 50 mM Tris-HCl,1 mM DTT, 0.1 mm EDTA, 50% glycerol, 0.1% Tritron X-100, pH 7.5) StorageStore all components at –20°C. Storage at –70°C may result in precipitation and loss of activity. Dilutions of rLysozyme Solution with rLysozyme Dilution Buffer of 1:100 or less are stable at 4°C for one week. Do not store diluted rLysozyme at –20°C because it will freeze and lose activityand/or precipitate.TB334 Rev. C 0503United States & Canada 800-207-0144Germany ***********United Kingdom 0800 622935Or your local sales office NovagenE. coli Lysis using Freeze/Thaw CycleThis protocol isolates the protein from the periplasm and cytoplasm using rLysozyme Solution orrLysozyme Solution, Veggie™ Grade and a freeze/thaw of the cell pellet. If a periplasmic fractionis desired, the osmotic shock procedure provided in the pET System Manual (Technical Bulletin055) should be followed. The final pellet from the osmotic shock procedure can then be used inthis protocol.1. Harvest cells from liquid culture by centrifugation at 10,000 × g for 10 min using a pre-weighed centrifuge tube. Decant and allow the pellet to drain, removing as much liquid aspossible. Determine the wet weight of the pellet.2. Freeze the pellet completely at –20°C or –70°C.3. Completely thaw the frozen cell pellet and resuspend in room temperature lysis buffer (50mM Tris-HCl or NaH2PO4, 50 mM NaCl, 5% glycerol, pH 7–8) using 7 ml lysis buffer per gramof wet cell paste and mix by pipetting up and down by by gentle vortexing. Althoughprotease inhibitors are often unnecessary, they can be added to protect against degradativeenzymes. Serine protease inhibitors should be avoided if the target protein is to be treatedwith Thrombin (Cat. No. 69671-3), Factor Xa (Cat. No. 69036-3) or RecombinantEnterokinase (rEK, Cat. No. 69066-3) because active inhibitor carried through thepurification may affect cleavage reactions. Include dialysis or gel filtration prior toproteolytic cleavage with rEK, Factor Xa or thrombin if serine protease inhibitors are used. Ifproteolytic degradation is a problem, try adding AEBSF (10–100 µM; Calbiochem Cat. No.101500), Pepstatin A (1 µM; Cat. No. 516482), Leupeptin (10–100 µM; Cat, No. 108975),Aprotinin (2 µg/ml; Cat. No. 616398), Benzamindine (15 µg/ml; Cat. No. 324890), or ProteaseInhibitor Cocktail Set II (with EDTA; Cat. No. 539132), or Set III (without EDTA; Cat. No.539134).Notes: DO NOT add rLysozyme Solution until a uniform cell suspension has been obtained. The freeze/thaw step ruptures the cell membrane allowing rLysozyme to access the cell wall. IfrLysozyme is added prematurely, the immediate viscosity increase will make complete cellresuspension difficult and incomplete lysis may result.rLysozyme Solution can be diluted with the rLysozyme Dilution Buffer as needed.Benzonase® Nuclease (Cat. No. 70664) degrades all forms of nucleic acid, eliminating viscosity and reducing processing time. 4. Add approximately 7.5 KU rLysozyme per 1 ml lysis buffer (45–60 KU/gram cell paste).5. Optional: Add 1 µl (25 units) Benzonase Nuclease per 1 ml lysis buffer used forresuspension. Benzonase is not recommended for nuclease free preparations. Proteinpurification may not remove Benzonase. Residual nuclease activity can be monitored by incubation of the purified protein with RNA or DNA markers followed by gel analysis. Other methods of reducing viscosity include shearing or precipitating the nucleic acids (2).6. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10–20 min at room temperature. The extract should not be viscous at the end of the incubation.Longer incubation time may be required if lysis is performed at 4ºC. Determine empirically.7. Remove insoluble debris by centrifugation at 16,000 × g for 20 min at 4°C.8. Transfer the supernatant to a fresh tube for analysis and/or purification. The soluble extractcan be loaded directly onto any Novagen protein purification resin (and numerous other systems). Maintain clarified extracts on ice for short-term storage (a few hours) or freeze at –20°C until needed.9. If desired, save the pellet for inclusion body purification. See Technical Bulletin 055 forrelevant protocols. Target proteins found in inclusion bodies that contain a His•Tag®sequence may be purified under denaturing conditions as described in Technical Bulletin 054. Inclusion bodies can be solubilized and refolded prior to purification (Technical Bulletin 234).TB334 Rev. C 0503United States & Canada 800-207-0144Germany ***********United Kingdom 0800 622935Or your local sales officeNovagenReferences1. Grabski, A., Mehler, M., Drott, D. and Van Dinther, J. (2002) in Nova tions14, 2–5.2. Burgess, R. R. (1991) Meth. Enzymol.208, 3–10.TB334 Rev. C 0503United States & Canada 800-207-0144 Germany *********** United Kingdom 0800 622935 Or your local sales officeNovagen。

BCA蛋白定量试剂盒使用说明书

BCA蛋白定量试剂盒使用说明书一、产品简介BCA 蛋白定量试剂盒是一种常用的蛋白质浓度测定方法，具有操作简便、灵敏度高、准确性好等优点。

本试剂盒适用于细胞裂解液、组织匀浆、血清、血浆等多种样品中总蛋白浓度的定量测定。

二、试剂盒组成1、 BCA 工作液：A 液与 B 液按 50:1 的比例混合而成，现配现用。

2、蛋白标准品（2mg/ml）：牛血清白蛋白（BSA）溶液。

3、 96 孔板三、所需设备1、酶标仪（波长 562nm）2、移液器（量程分别为20μl、200μl、1000μl）3、涡旋振荡器4、离心机四、操作步骤1、标准曲线的绘制（1）将蛋白标准品（2mg/ml）用 PBS 或生理盐水稀释成一系列浓度梯度，如0μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml、600μg/ml、800μg/ml、1000μg/ml、1500μg/ml、2000μg/ml。

（2）在 96 孔板中，每个浓度梯度设置 2 个复孔。

分别向孔中加入25μl 不同浓度的标准品溶液。

（3）向每个孔中加入200μl BCA 工作液，轻轻振荡混匀，37℃孵育 30 分钟。

（4）使用酶标仪在 562nm 波长下测定吸光度值（OD 值）。

（5）以蛋白浓度为横坐标，OD 值为纵坐标，绘制标准曲线。

2、样品测定（1）将待测样品用 PBS 或生理盐水适当稀释（若样品浓度过高，可能会超出标准曲线范围）。

（2）在 96 孔板中，设置样品孔和空白孔（仅加25μl 稀释液和200μl BCA 工作液）。

向样品孔中加入25μl 稀释后的样品。

（3）向每个孔中加入200μl BCA 工作液，轻轻振荡混匀，37℃孵育 30 分钟。

（4）使用酶标仪在 562nm 波长下测定样品孔和空白孔的吸光度值（OD 值）。

（5）样品的蛋白浓度可根据标准曲线计算得出。

五、注意事项1、 BCA 工作液应现配现用，避免长时间放置导致失效。

1911 蛋白酶K

使用方法：根据实验需要使用，用纯水稀释。
相关产品：
产品总蛋白提取试剂盒核蛋白提取试剂盒膜/胞浆/核蛋白分步提取试剂盒
产品号 BB-3101 BB-3102 BB-3104
产品磷酸化蛋白富集试剂盒膜蛋白提取试剂盒活性蛋白提取试剂盒
产品号 BB-3108 BB-3103 BB-3106

400-8363-211
本产品仅供科学研究使用！请勿用于临床1 BB-3121 BB-3122 BB-3123 BB-3125 BB-3126 BB-3105 BB-3181 BB-3183 BB-3187
产品说明书
BCA 蛋白定量试剂盒植物核蛋白提取试剂盒细菌膜蛋白提取试剂盒植物总蛋白提取试剂盒植物膜蛋白提取试剂盒蛋白酶抑制剂混合物真菌蛋白提取试剂盒磷酸酶抑制剂混合物细菌蛋白提取盒（2D 电泳用）酵母蛋白提取盒（2D 电泳用）线粒体蛋白提取盒（2D 电泳用）
蛋白酶 K（20mg/ml）
货号：BB-1911
试剂盒储存条件： -20℃保存。
试剂盒组成：
产品组成规格
蛋白酶 K（20mg/ml）使用说明书
有效期：一年。
BB-1911-1 1ml 1ml 1
BB-1911-2 5ml 1ml*5 1
产品说明书 V2.16
组份编号 31010A
※ ※ ※ 使用前请仔细阅读产品说明书 ※ ※ ※

-1-
咨询邮箱：bestbio@ 电话：021-33921235
400-8363-211
本产品仅供科学研究使用！请勿用于临床、诊断、食品、化妆品检测等用途！
产品说明书
● 样品或试剂被细菌或真菌污染或试剂交叉污染可能会导致错误的结果。 ● 最好使用一次性吸头、管、瓶或玻璃器皿，可重复使用的玻璃器皿必须在使用前清

Solutol HS-15_AbMole中国_CAS号61909-81-7_M9380说明书Protocol

产品说明Protocol分子量溶解性（25°C ）DMSO N/A 分子式(C2H4O)nC18H36O3Water ≥ 15 mg/mL CAS 号61909-81-7Ethanol Soluble储存条件4°C, dry, sealed生物活性Solutol HS-15 是一种非离子型增溶剂，应用在亲脂性药物的注射剂、乳剂、脂质制剂中。

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA 指南）小鼠大鼠兔豚鼠仓鼠狗重量 (kg)0.020.15 1.80.40.0810体表面积 (m )0.0070.0250.150.050.020.5K 系数36128520动物A (mg/kg) = 动物B (mg/kg) ×动物B 的K 系数动物A 的K 系数例如，依据体表面积折算法，将白藜芦醇用于小鼠的剂量22.4 mg/kg 换算成大鼠的剂量，需要将22.4 mg/kg 乘以小鼠的K 系数（3），再除以大鼠的K 系数（6），得到白藜芦醇用于大鼠的等效剂量为11.2 mg/kg 。

参考文献Mechanism of mucosal permeability enhancement of CriticalSorb (Solutol HS15) investigated in vitro in cell cultures.Shubber S, et al . Pharm Res. 2015 Feb;32(2):516-27. PMID: 25190006.抑制剂化合物库CCK-8 / 蛋白酶抑制剂CocktailAbMole 中国Solutol HS-15 目录号M9380化学数据所有产品仅供科研使用，严禁用于人或动物的治疗等任何其他用途，不为任何个人提供产品和服务。

2m m m m m。

常见蛋白酶抑制剂

之巴公井开创作当前位置：生物帮> 实验技巧> 生物化学技术> 正文蛋白酶及蛋白酶抑制剂大全日期：2012-06-13 来源：互联网标签：相关专题：解析蛋白酶活性测定聚焦蛋白酶研究新进展摘要 : 破碎细胞提取蛋白质的同时可释放出蛋白酶，这些蛋白酶需要迅速的被抑制以坚持蛋白质不被降解。

在蛋白质提取过程中，需要加入蛋白酶抑制剂以防止蛋白水解。

以下列举了5种经常使用的蛋白酶抑制剂和他们各自的作用特点，因为各种蛋白酶对分歧蛋白质的敏感性各不相同，因此需要调整各种蛋白酶的浓度恩必美生物新一轮2-5折生物试剂大促销！Ibidi细胞灌流培养系统-模拟血管血液流动状态下的细胞培养系统广州赛诚生物基因表达调控专题蛋白酶抑制剂破碎细胞提取蛋白质的同时可释放出蛋白酶，这些蛋白酶需要迅速的被抑制以坚持蛋白质不被降解。

在蛋白质提取过程中，需要加入蛋白酶抑制剂以防止蛋白水解。

以下列举了5种经常使用的蛋白酶抑制剂和他们各自的作用特点，因为各种蛋白酶对分歧蛋白质的敏感性各不相同，因此需要调整各种蛋白酶的浓度。

由于蛋白酶抑制剂在液体中的溶解度极低，尤其应注意在缓冲液中加人蛋白酶抑制剂时应充分混匀以减少蛋白酶抑制剂的沉淀。

在宝灵曼公司的目录上可查到更完整的蛋白酶和蛋白酶抑制剂表。

经常使用抑制剂PMSF1)抑制丝氨酸蛋白酶(如胰凝乳蛋白酶，胰蛋白酶，凝血酶)和巯基蛋白酶(如木瓜蛋白酶);2)10mg/ml溶于异丙醇中;3)在室温下可保管一年;4)工作浓度:17~174ug/ml(0.1~1.0mmol/L);5)在水液体溶液中不稳定，必须在每一分离和纯化步调中加入新鲜的PMSF。

EDTA1)抑制金属蛋白水解酶;2)0.5mol/L水溶液，pH8~9;3)溶液在4℃稳定六个月以上;4)工作浓度:0.5~1.5mmol/L. (0.2~0.5mg/ml);5)加入NaOH调节溶液的pH值，否则EDTA不溶解。

胃蛋白酶抑制剂(pepstantin)l)抑制酸性蛋白酶如胃蛋白酶，血管紧张肽原酶，组织蛋白酶D和凝乳酶;2)1mg/ml溶于甲醇中;3}储存液在4℃一周内稳定，-20℃稳定6个月;4)1作浓度:0.7ug/ml(1umol/L)5)在水中不溶解。

cusabio 微量白蛋白尿(MAU ALB)检测试剂盒使用说明书

Human microalbunminuria(MAU/ALB) ELISA kit Catalog Number. CSB-E08970hFor the quantitative determination of human microalbunminuria(MAU/ALB) concentrations in serum, plasma, urine.This package insert must be read in its entirety before using this product.If You Have ProblemsTechnical Service Contact informationPhone: 86-27-87582341Fax: 86-27-87196150Email:****************Web: In order to obtain higher efficiency service, please ready to supply the lot numberof the kit to us (found on the outside of the box).1PRINCIPLE OF THE ASSAYThis assay employs the competitive inhibition enzyme immunoassay technique. Antibody specific for MAU has been pre-coated onto a microplate. Standards and samples are pipetted into the wells with biotin-conjugated MAU. A competitive inhibition reaction is launched between MAU (Standards or samples) and biotin-conjugated MAU with the pre-coated MAU antibody. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in opposite to the amount of MAU bound in the initial step. The color development is stopped and the intensity of the color is measured.DETECTION RANGE0.078 µg/ml-5 µg/ml.SENSITIVITYThe minimum detectable dose of human MAU is typically less than 0.019 µg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest human MAU concentration that could be differentiated from zero.SPECIFICITYThis assay has high sensitivity and excellent specificity for detection of human MAU. No significant cross-reactivity or interference between human MAU and analogues was observed.Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between human MAU and all the analogues, therefore, cross reaction may still exist.2PRECISIONIntra-assay Precision (Precision within an assay): CV%<8%Three samples of known concentration were tested twenty times on one plate to assess.Inter-assay Precision (Precision between assays):CV%<10%Three samples of known concentration were tested in twenty assays to assess.LIMITATIONS OF THE PROCEDUREFOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.3MATERIALS PROVIDEDReagents QuantityAssay plate (12 x 8 coated Microwells) 1(96 wells) Standard (Freeze dried) 2Biotin-conjugate (100 x concentrate) 1 x 60 µlHRP-avidin (100 x concentrate) 1 x 120 µlBiotin-conjugate Diluent 1 x 10 mlHRP-avidin Diluent 1 x 20 ml Sample Diluent 2 x 20 mlWash Buffer (25 x concentrate) 1 x 20 mlTMB Substrate 1 x 10 mlStop Solution 1 x 10 ml Adhesive Strip (For 96 wells) 4Instruction manual 1STORAGEUnopenedkitStore at 2 - 8°C. Do not use the kit beyond the expiration date.Opened kitCoated assayplateMay be stored for up to 1 month at 2 - 8°C.Try to keep it in a sealed aluminum foil bag,and avoid the damp.Standard May be stored for up to 1 month at 2 - 8° C.If don’t make recent use, better keep it storeat -20°C.HRP-avidinBiotin-conjugateBiotin-conjugateDiluentMay be stored for up to 1 month at 2 - 8°C. HRP-avidinDiluentSample DiluentWash BufferTMB SubstrateStop Solution*Provided this is within the expiration date of the kit.4OTHER SUPPLIES REQUIREDMicroplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.An incubator which can provide stable incubation conditions up to 37°C±0.5°C.Squirt bottle, manifold dispenser, or automated microplate washer.Absorbent paper for blotting the microtiter plate.100ml and 500ml graduated cylinders.Deionized or distilled water.Pipettes and pipette tips.Test tubes for dilution.PRECAUTIONSThe Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.5SAMPLE COLLECTION AND STORAGESerum Use a serum separator tube (SST) and allow samples to clot for30 minutes before centrifugation for 15 minutes at 1000 x g, 2 - 8°C.Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay.Plasma Collect plasma using EDTA, or heparin as an anticoagulant.Centrifuge for 15 minutes at 1000 x g, 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay.Urine Use a sterile container to collect urine samples. Remove any particulates by centrifugation for 15 minutes at 1000xg, 2 - 8°C and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge again before assaying to remove any additional precipitates that may appear after storage.SAMPLE PREPARATIONRecommend to dilute the serum or plasma samples 50000-fold before test.The suggested 50000-fold dilution can be achieved by adding 2µl sample to 398µl of normal saline. Complete the 50000-fold dilution by adding 2µl of this solution to 498µl of Sample Diluent. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments.Recommend to dilute the urine samples with Sample Diluent(1:40) before test. The suggested 40-fold dilution can be achieved by adding 6µl sample to 234µl of Sample Diluent. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments6Note:1. CUSABIO is only responsible for the kit itself, but not for the samplesconsumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.2. Samples to be used within 5 days may be stored at 2-8°C, otherwisesamples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid loss of bioactivity and contamination.3. Grossly hemolyzed samples are not suitable for use in this assay.4. If the samples are not indicated in the manual, a preliminary experiment todetermine the validity of the kit is necessary.5. Please predict the concentration before assaying. If values for these arenot within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.6. Tissue or cell extraction samples prepared by chemical lysis buffer maycause unexpected ELISA results due to the impacts of certain chemicals.7. Owing to the possibility of mismatching between antigen from otherresource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.8. Influenced by the factors including cell viability, cell number and alsosampling time, samples from cell culture supernatant may not be detected by the kit.9. Fresh samples without long time storage are recommended for the test.Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.7REAGENT PREPARATIONNote:Kindly use graduated containers to prepare the reagent. Please don't prepare the reagent directly in the Diluent vials provided in the kit. Bring all reagents to room temperature (18-25°C) before use for 30min.Prepare fresh standard for each assay. Use within 4 hours and discard after use.Making serial dilution in the wells directly is not permitted.Please carefully reconstitute Standards according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved.To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10µl for once pipetting.Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result.1. Biotin-conjugate (1x) - Centrifuge the vial before opening.Biotin-conjugate requires a 100-fold dilution. A suggested 100-fold dilution is 10 µl of Biotin-conjugate + 990 µl of Biotin-conjugate Diluent.2. HRP-avidin (1x) - Centrifuge the vial before opening.HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 µl of HRP-avidin + 990 µl of HRP-avidin Diluent.3. Wash Buffer(1x)- If crystals have formed in the concentrate, warm up toroom temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 ml of Wash Buffer (1 x).894.StandardCentrifuge the standard vial at 6000-10000rpm for 30s.Reconstitute the Standard with 1.0 ml of Sample Diluent . Do not substitute other diluents. This reconstitution produces a stock solution of 5 µg/ml. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.Pipette 150 µl of Sample Diluent into each tube (S0-S6). Use the stock solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard (5 µg/ml). Sample Diluent serves as the zero standard (0 µg/ml).Tube S7 S6 S5S4 S3 S2 S1 S0 µg/ml52.51.250.6250.3120.1560.078ASSAY PROCEDUREBring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay.It is recommended that all samples and standards be assayed in duplicate.1. Prepare all reagents, working standards, and samples as directed in theprevious sections.2. Refer to the Assay Layout Sheet to determine the number of wells to beused and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.3. Set a Blank well without any solution.4. Add 50µl of standard and sample per well.5. Add 50µl of Biotin-conjugate(1x) to each well(not to Blank well). Coverwith a new adhesive strip. Incubate for 60 minutes at 37°C.(Biotin-conjugate(1x) may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)6. Aspirate each well and wash, repeating the process two times for a total ofthree washes. Wash by filling each well with Wash Buffer (200µl) using a squirt bottle, multi-channel pipette, manifold dispenser, or autowasher, and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.7. Add 100µl of HRP-avidin(1x) to each well(not to Blank well). Cover themicrotiter plate with a new adhesive strip. Incubate for 60 minutes at 37°C.8. Repeat the aspiration/wash process for five times as in step 6.9. Add 90µl of TMB Substrate to each well. Incubate for 20 minutes at 37°C.Protect from light.10. Add 50µl of Stop Solution to each well, gently tap the plate to ensurethorough mixing.1011. Determine the optical density of each well within 5 minutes, using amicroplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.*Samples may require dilution. Please refer to Sample Preparation section. Note:1. The final experimental results will be closely related to validity of theproducts, operation skills of the end users and the experimental environments.2. Samples or reagents addition: Please use the freshly prepared Standard.Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions.Also, use separate reservoirs for each reagent.3. Incubation: To ensure accurate results, proper adhesion of plate sealersduring incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed.4. Washing: The wash procedure is critical. Complete removal of liquid ateach step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and falsely elevated absorbance reading. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.115. Controlling of reaction time: Observe the change of color after adding TMBSubstrate (e.g. observation once every 10 minutes), TMB Substrate should change from colorless or light blue to gradations of blue. If the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.6. TMB Substrate is easily contaminated. TMB Substrate should remaincolorless or light blue until added to the plate. Please protect it from light.7. Stop Solution should be added to the plate in the same order as the TMBSubstrate. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.1213ASSAY PROCEDURE SUMMARY*Samples may require dilution. Please refer to Sample Preparation section.CALCULATION OF RESULTSUsing the professional soft "Curve Expert" to make a standard curve is recommended, which can be downloaded from our web.Average the duplicate readings for each standard and sample and subtract the average optical density of Blank.Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the MAU concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.14人尿微量白蛋白(MAU/ALB)酶联免疫试剂盒使用说明书【产品编号】CSB-E08970h【预期应用】ELISA法定量测定人血清、血浆、尿液中MAU含量。

罗氏蛋白酶抑制剂说明

Pancreasextract
0.02
7.8 7.8
Thermolysin 0.0005 (Metalloprotease) Chymotrypsin Trypsin Papain 0.002 0.02 0.02 0.002 0.33
2.
How 7.8 7.8 7.8
For the complete inhibition of serine and cysteine proteases during extractions from animal and plant tissues or cells, yeast and bacteria
Cat. No. 04 693 159 001
0113.04728688001➂
Table 1: Inhibition of different proteases by cOmplete Protease Inhibitor Tablets. One cOmplete, EDTA-free tablet was added per 10 ml incubation solution. Proteolytic activity was determined with the Roche Applied Science Universal Protease Substrate (casein, resorufin-labeled*). When extractions or single-step isolations are necessary in the acid pH range, simply include pepstatin* along with cOmplete, EDTA-free tablets to ensure aspartic (acid) protease inhibition. All experiments were performed at room temperature.

FITC-Dextran_AbMole中国_CAS号60842-46-8_M9390说明书Protocol

产品说明Protocol分子量溶解性（25°C ）DMSO N/A 分子式Water ≥ 30 mg/mL CAS 号60842-46-8Ethanol N/A储存条件4°C, protect from light, dry, sealed生物活性FITC-Dextran 是一种由FITC 偶联葡聚糖形成的标记物。

参考文献Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells.Eriksson I, et al . Methods Mol Biol. 2017;1594:179-189. PMID: 28456983.Fluorescein Isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability.Natarajan R, et al . Curr Protoc Neurosci. 2017 Apr 10;79:9.58.1-9.58.15. PMID: 28398646.High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations.Hoffmann A, et al . Transl Stroke Res. 2011 Mar;2011 Mar. PMID: 21423333.抑制剂化合物库CCK-8 / 蛋白酶抑制剂CocktailAbMole 中国FITC-Dextran 目录号M9390化学数据所有产品仅供科研使用，严禁用于人或动物的治疗等任何其他用途，不为任何个人提供产品和服务。

蛋白酶抑制剂的配制

How to make protease and phosphotase inhibitors Protease inhibitors are rapidly inactivated in aqueous solutions and should be added to solutions immediately before use. Cocktail solutions are stored at -20 degrees and are good for approximately 6 months.I. Protease Inhibitor CocktailThe Protease inhibitor cocktail is used as 100X for extract preps and as 1000X for column elution buffers. Cocktail is made in 100% EtOH and contains:Some components are not completely soluble in 100% EtOH. Vortex solution well before use to obtain a cloudy suspension and add to buffer - they become soluble in aqueousII. 100 mM PMSFPMSF is very unstable in aqueous solutions. The 100 mM stock solution is made in isopropanol and stored in the dark at -20 degrees. Must be warmed (37 degrees) to get into solution. Use at 1 mM in solutions.Activation of Sodium Orthovanadate (sigma s-6508)Sodium orthovanadate should be activated for maxima l inhibition of protein phosphotyrosyl-phosphatases .1. Prepare powder needed for 200 mM solution of sodium orthovanadate,dissolve in ddH2O.2. Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH ofthe sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow.3. Boil the solution until it turns colorless (approximately 10 minutes).4. Cool to room temperature.5. Readjust the pH to 10.0 and repeat steps 3 and 4 until the solutionremains colorless and the pH stabilizes at 10.0.6. adjust volume.7. Store the activated sodium orthovanadate as aliquots at -20°C.This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of protein tyrosine phosphatases. Please note that adding DTT rapidly inactivates sodium orthovanadate.Sodium fluoride (Sigma, s-1504)Dissolve sodium fluoride to 500mM in ddH20, store in darkness at 4 degree. Also you first make activated 400 mM sodium orthovanadata and 1 M sodium fluoride separately, and then 1:1 mix them together to a 100x concentration (200mM sodium orthovanadata and 500 mM sodium fluoride). Aliquot to 15ml tubes, keep in -20°C and in darkness.。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

奥默生物电话：021-5096 7598 邮箱：techsupport@ 网站：
产品简介：
本产品是由6种独立的蛋白酶抑制剂（不含EDTA）按照优化的配比配置而成的蛋白酶抑制剂混合物。

其作用的靶点分别为丝氨酸蛋白酶，丝氨酸半胱氨酸蛋白酶，氨肽酶，半胱氨酸蛋白酶和天冬氨酸蛋白酶等，广泛作用于各种蛋白酶，可称之为广谱蛋白酶抑制剂。

本产品应于-20℃环境下密封保存，产品有效期为2年。

使用时可短时间放置于常温或4℃中，用毕应立即放回-20℃中保存。

储存条件：
蛋白酶抑制剂Cocktail (不含EDTA，100X DMSO储液)
蛋白酶抑制剂Cocktail 使用说明书
蛋白酶抑制剂通过抑制蛋白酶的活性，减少蛋白酶对蛋白的分解，从而有效地提高蛋白质得出率，而且不使细胞及组织本身的性质发生改变。

作用原理：
产品组成：成分
MTT法CCK法 AEBSF
Aprotinin
Bestatin
E-64
Leupeptin
Pepstatin A Cysteine proteases Serine proteases Aminopeptidases Serine proteases Serine and cysteine proteases Aspartic proteases Irreversible Reversible Reversible Reversible
Reversible Irreversible 靶点靶点类型运输条件：蓝冰运输。

奥默生物
使用说明：
1. 本产品适用于哺乳动物细胞及组织的蛋白质提取及纯化，蛋白免疫印迹（WesternBlot）,
免疫共沉淀（Co-IP），免疫荧光（IF），免疫组织化学（IHC），激酶测定（kinaseassay）
和抗体，酶诊断试剂盒（Dignose Kit）等。

2. 实验时，按照1:100的容积比，将Cocktail预先加入在已准备好的实验体系中，轻轻混合均
匀。

注意事项：
1. 本产品不含EDTA，为100×DMSO储存液形式。

使用时应调整Cocktail浓度为1×，若有蛋
白酶含量丰富的细胞或组织，可适当增加Cocktail在实验体系中的浓度。

2. 本产品置于-20℃保存时，DMSO呈现冰晶状态，此属于正常现象，非产品质量问题。

3. 本产品仅供科研使用。

常见问题解答：
1. 蛋白酶抑制剂Cocktail有哪些优势？
回答：针对多种氨基酸的靶点，对目的蛋白进行全方位的保护，使其免受内源性蛋白酶的降
解，蛋白的得出率较高，大大提高实验效率。

2. 可以配合细胞裂解液一起使用吗？
回答：可以，这样可以充分的裂解细胞或组织中的蛋白，有效降低蛋白质的降解。

3. 为什么需要用DMSO溶解？可否换成其他溶剂？
回答：蛋白酶抑制剂Cocktail较易溶解于DMSO，且DMSO通常不会影响实验结果，而且性
质稳定，利于保存和运输。

因此暂不考虑其他溶剂。

4. 实验时误将其放置常温下，会不会造成试剂变质？
回答：短时间放置于常温下是可以的，不会造成试剂变质，因为蛋白酶抑制剂通常比较稳定。

如果实验需要在常温下进行，建议将其置于冰块操作，用毕后迅速放置-20℃保存。

同时应尽
量避免反复冻融。

电话：021-5096 7598 邮箱：techsupport@ 网站：。