Enoxacin_74011-58-8_DataSheet_MedChemExpress
HSF1A_DataSheet_MedChemExpress
Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:HSF1A is a cell–permeable activator of heat shock transcription factor 1 (HSF1).IC50 & Target: HSF1[1]In Vitro: HSF1A protects cells from stress–induced apoptosis, binds TRiC subunits and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. Moreover, fluorescence anisotropy experiments using FITC coupled to HSF1A demonstrates that HSF1A–FITC binds to a purified Tcp1 subunit of TRiC with an affinity of approximately 600 nM. This is validated qualitatively via titration of purified Tcp1 into binding reactions containing 500 nM Biotin or HSF1A–Biotin [1]. Quantification bycounting the number of cell containing aggregates as a function of the total number of cells reveals that at HSF1A concentrations as low as 2 μM, a reduced number of aggregate–containing cells are observed. The fraction of cells containing aggregates continued to decrease in a dose–dependent manner such that pretreatment with 12 μM HSF1A resulta in ~20% of the cells exhibiting aggregates visible by fluorescence microscopy [2].In Vivo: HSF1A enhances HSF1 activity, stabilizes HSF1 expression and minimizes Doxorubicin (DOX)–induced cardiac damage. WKY rats are challenged with DOX (accumulated dose: 30 mg/kgw), and DOX combined with HSF1A (100 mg/kgw/day). Supplementation with HSF1A significantly elevates cardiac functions back to the levels of the control group. HSF1A has been shown to stimulate human HSF1 nuclear translocation, elevate protein chaperone expression and ameliorate protein misfolding and cell death in aneurodegenerative disease model. The echocardiographic results show that HSF1A also alleviates DOX–induced failures in cardiac function [3].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[1]Protein extracts are generated from mammalian, yeast and E. coli cultures using biotin–binding buffer (20 mM HEPES,5 mM MgCl 2, 1 mM EDTA, 100 mM KCl, 0.03% NP–40) supplemented with 1% Trition–X100 and protease inhibitors. Approximately 0.5mg of protein extract is incubated with 100 μM HSF1A–Biotin for 4 h at 4°C and HSF1A–Biotin associated proteins captured by with NeutrAvidin Agarose Resin. After washing in biotin binding buffer proteins are eluted using 50 μL biotin elution buffer (100 mM Tris,150 mM NaCl, 0.1 mM EDTA, 2 mM D–biotin), resolved on a 4–20% SDS–PAGE, and immunoblotted. For purified TRiC and Hsp70analyses, 5 nM protein is incubated in biotin–binding buffer+0.5% Triton X–100 with 100 μM biotin or 100 μM HSF1A–Biotin for 4 h at 4°C and captured with NeutrAvidin Resin. For NiNTA purified yeast Tcp1, different concentrations of Tcp1 0.5 μM, 1 mM, 2 mM, 3 mM and 4 mM in 25 mM Hepes pH 7.5, 150 mM NaCl are incubated with 0.5 μM Biotin or HSF1A–Biotin for 4 h at 4°C and captured with NeutrAvidin Resin [1].Cell Assay:[2]PC12 cells seeded into a 96–well plate (5×104 cells/well) are treated with increasing concentrations of HSF1A (2, 4, 8 andProduct Name:HSF1A Cat. No.:HY-103000CAS No.:1196723-93-9Molecular Formula:C21H19N3O2S2Molecular Weight:409.52Target:HSP Pathway:Cell Cycle/DNA Damage; Metabolic Enzyme/Protease Solubility:DMSO: ≥ 150 mg/mL12 μM) for 15 h, at which time httQ74–GFP expression is stimulated by incubation in the presence of 1 μg/mL Doxycycline for 5 d. Cell viability is assessed via the XTT viability assay[2].Animal Administration:[3]Rat[3]Ten–week–old Wistar Kyoto rats (WKY) are used. The rats are housed at a constant temperature (22°C) on a 12–h light/dark cycle with food and tap water. The animals are arranged into three groups: WKY rats (the control group), DOX rats and DOX rats treated with HSF1A. Each group contain five animals. The DOX group is injected with DOX (5 mg/kg) for 6 consecutive weeks intraperitoneal injection to achieve a cumulative dose of 30 mg/kg, which has been well documented to achieve cardiotoxicity. The small molecular HSF1 activator HSF1A (100 mg/kg/day) is injected intraperitoneally.References:[1]. Neef DW, et al. A direct regulatory interaction between chaperonin TRiC and stress–responsive transcription factor HSF1. Cell Rep. 2014 Nov 6;9(3):955–66.[2]. Neef DW, et al. Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease. PLoS Biol. 2010 Jan 19;8(1):e1000291.[3]. Huang CY, et al. Doxorubicin attenuates CHIP–guarded HSF1 nuclear translocation and protein stability to trigger IGF–IIR–dependent cardiomyocyte death. Cell Death Dis. 2016 Nov 3;7(11):e2455.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。
转基因植物NOS基因核酸检测试剂盒(PCR-荧光探针法)说明书
转基因植物NOS基因核酸检测试剂盒(PCR-荧光探针法)说明书转基因植物NOS基因核酸检测试剂盒(PCR荧光探针法)说明书试剂盒简介货为了适应新加坡石斑鱼虹彩病毒染料法荧光定量PCR试剂盒快速检测和疫病研究的需要，本公司参照 OIE国际标准中规定的引物序列，经多次实验及系统优化，开发生产了本试剂盒。
应用本试剂盒进行检测具有快速、灵敏、特异、准确、安全操作简单、应用广泛和高通量检测等特点及优点。
试剂盒组成及试剂配制：酶联板（Assay plate）：一块（96孔）。
2.标准品（Standard）：2瓶（冻干品）。
3.样品稀释液（Sample Diluent）：1×20ml/瓶4.生物su标记抗体稀释液（Biotinantibody Diluent）：1×10ml/瓶。
5.辣根过氧化物酶标记亲和素稀释液（HRPavidin Diluent）：1×10ml/瓶。
6.生物su标记抗体（Biotinantibody）：1×120μl/瓶（1：100）7.辣根过氧化物酶标记亲和素（HRPavidin）：1×120μl/瓶（1：100）8.底物溶液（TMB Substrate）：1×10ml/瓶。
9.浓洗涤液（Wash Buffer）：1×20ml/瓶，使用时每瓶用蒸馏水稀释25倍。
10.终止液（Stop Solution）：1×10ml/瓶（2N H2SO4）。
样本处理及要求：1.血清：全血标本请于室温放置2小时或4℃过夜后于1000g离心20分钟，取上清即可检测，或将标本放于20℃或80℃保存，但应避免反复冻融。
2. 血浆：可用EDTA或肝素作为抗凝剂，标本采集后30分钟内于2 8°C 1000g离心20分钟，或将标本放于20℃或80℃保存，但应避免反复冻融。
3. 组织匀浆：用预冷的PBS (0.01M, pH=7.4)冲洗组织，去除残留血液（匀浆中裂解的红细胞会影响测量结果），称重后将组织剪碎。
微生物CLSI文件集锦（你想要的都在这里）
微生物CLSI文件集锦（你想要的都在这里）说起临床微生物的CLSI文件，大家首先想到的就是CLSI M100S——《抗微生物药物敏感性试验的执行标准》。
但是其实，与临床微生物相关的CLSI文件很多，截至2017年7月，已多达43个。
下面，小编就给您简单介绍一下吧。
1、M02-A12:Approved Standard中文：抗菌药敏试验的性能标准英文：Performance Standards for Antimicrobial Disk Susceptibility Tests内容与解释：介绍药物纸片扩散法的质量控制标准和最新折点标准。
2、M06-A2: Approved Standard中文：脱水MH琼脂的评估程序英文：Protocols for Evaluating Dehydrated Mueller-Hinton Agar内容与解释：略3、M07-A10:Approved Standard中文：需氧菌稀释法抗菌药物敏感性试验英文：Methods for Dilution Antimicrobial Susceptibility T ests for Bacteria That Grow Aerobically内容与解释：描述了肉汤稀释法和琼脂稀释法，而且还包含这些方法的标准化操作流程和CLSI推荐方法的性能，局限性，适应性。
4、M11-A8:Approved Standard中文：厌氧菌抗菌药物敏感性试验英文：Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria内容与解释：在过去的几年内，大部分厌氧菌的耐药表型都发生了很大的改变，导致了许多菌种的经验用药的面临很大的挑战。
对于厌氧菌，琼脂稀释法仍然是参考方法，对于调查研究和科研同样都适用。
而且其他的方法的对比标准也进行了说明，肉汤稀释法也应用于临床实验室，但是现在对脆弱拟杆菌和一些抗生素没有标准。
酶联免疫分析试剂盒说明书
人chemerin酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用预期应用ELISA法定量测定人血清、血浆或其它相关液体中chemerin含量。
实验原理本试剂盒应用双抗体夹心酶标免疫分析法测定标本中chemerin水平。
用纯化的抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入chemerin抗原、生物素化的抗人chemerin抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。
TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的chemerin呈正相关。
用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
试剂盒组成及试剂配制1.酶联板：一块（96孔）2.标准品（冻干品）：2瓶，每瓶临用前以样品稀释液稀释至1ml，盖好后静置10分钟以上，然后反复颠倒/搓动以助溶解，其浓度为20,000pg/ml，做系列倍比稀释后，分别稀释10,000pg/ml，5,000pg/ml，2,500pg/ml，1,250pg/ml，625pg/ml，312.5pg/ml，156pg/ml，样品稀释液直接作为标准浓度0pg/ml，临用前15分钟内配制。
如配制10,000pg/ml标准品：取0.5ml20,000pg/ml的上述标准品加入含有0.5ml样品稀释液的Eppendorf管中，混匀即可，其余浓度以此类推。
3.样品稀释液：1×20ml/瓶。
4.检测稀释液A：1×10ml/瓶。
5.检测稀释液B：1×10ml/瓶。
6.检测溶液A：1×120ul/瓶（1:100）临用前以检测稀释液A1:100稀释，稀释前根据预先计算好的每次实验所需的总量配制（每孔100ul），实际配制时应多配制0.1-0.2ml。
如1ul检测溶液A加99ul检测稀释液A的比例配制，轻轻混匀，在使用前一小时内配制。
7.检测溶液B：1×120ul/瓶（1:100）临用前以检测稀释液B1:100稀释。
稳定性英文版
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFLUOXETINE HClC17H18F3NO•HClM.W. = 345.79CAS — 59333-67-4STABILITY INDICATINGA S S A Y V A L I D A T I O NMethod is suitable for:ýIn-process controlþProduct ReleaseþStability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANY MECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationTABLE OF CONTENTS INTRODUCTION........................................................................................................................ PRECISION............................................................................................................................... System Repeatability ................................................................................................................ Method Repeatability................................................................................................................. Intermediate Precision .............................................................................................................. LINEARITY................................................................................................................................ RANGE...................................................................................................................................... ACCURACY............................................................................................................................... Accuracy of Standard Injections................................................................................................ Accuracy of the Drug Product.................................................................................................... VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION........................................... Linearity at Low Concentrations................................................................................................. Accuracy of Fluoxetine HCl at Low Concentration..................................................................... System Repeatability................................................................................................................. Quantitation Limit....................................................................................................................... Detection Limit........................................................................................................................... VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES).................................. Meta-Fluoxetine HCl linearity at 0.05% - 1.0%........................................................................... Detection Limit for Fluoxetine HCl.............................................................................................. Quantitation Limit for Meta Fluoxetine HCl................................................................................ Accuracy for Meta-Fluoxetine HCl ............................................................................................ Method Repeatability for Meta-Fluoxetine HCl........................................................................... Intermediate Precision for Meta-Fluoxetine HCl......................................................................... SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD............................. FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD..................................1. Unstressed analysis...............................................................................................................2. Acid Hydrolysis stressed analysis..........................................................................................3. Base hydrolysis stressed analysis.........................................................................................4. Oxidation stressed analysis...................................................................................................5. Sunlight stressed analysis.....................................................................................................6. Heat of solution stressed analysis.........................................................................................7. Heat of powder stressed analysis.......................................................................................... System Suitability stressed analysis.......................................................................................... Placebo...................................................................................................................................... STABILITY OF STANDARD AND SAMPLE SOLUTIONS......................................................... Standard Solution...................................................................................................................... Sample Solutions....................................................................................................................... ROBUSTNESS.......................................................................................................................... Extraction................................................................................................................................... Factorial Design......................................................................................................................... CONCLUSION...........................................................................................................................ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationBACKGROUNDTherapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptake inhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloride has been shown to have comparable efficacy to tricyclic antidepressants but with fewer anticholinergic side effects. The patent expiry becomes effective in 2001 (US). INTRODUCTIONFluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosage strengths with the same capsule weight. The formulas are essentially similar and geometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20 mg strength.The following validation, for the method SI-IAG-206-02 , includes assay and determination of Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06. Currently the method is the in-house method performed for Stability Studies. The Validation was performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.In the forced degradation studies, the two placebo samples were also used. PRECISIONSYSTEM REPEATABILITYFive replicate injections of the standard solution at the concentration of 0.4242mg/mL as described in method SI-IAG-206-02 were made and the relative standard deviation (RSD) of the peak areas was calculated.SAMPLE PEAK AREA#15390#25406#35405#45405#55406Average5402.7SD 6.1% RSD0.1ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::PRECISION - Method RepeatabilityThe full HPLC method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method repeated six times and the relative standard deviation (RSD) was calculated.SAMPLENumber%ASSAYof labeled amountI 96.9II 97.8III 98.2IV 97.4V 97.7VI 98.5(%) Average97.7SD 0.6(%) RSD0.6PRECISION - Intermediate PrecisionThe full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method was repeated six times by a second analyst on a different day using a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.SAMPLENumber% ASSAYof labeled amountI 98.3II 96.3III 94.6IV 96.3V 97.8VI 93.3Average (%)96.1SD 2.0RSD (%)2.1The difference between the average results of method repeatability and the intermediate precision is 1.7%.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationLINEARITYStandard solutions were prepared at 50% to 200% of the nominal concentration required by the assay procedure. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over the concentration range required. Y-Intercept was found to be insignificant.RANGEDifferent concentrations of the sample (IAG-21-001) for the 20mg dosage form were prepared, covering between 50% - 200% of the nominal weight of the sample.Conc. (%)Conc. (mg/mL)Peak Area% Assayof labeled amount500.20116235096.7700.27935334099.21000.39734463296.61500.64480757797.52000.79448939497.9(%) Average97.6SD 1.0(%) RSD 1.0ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::RANGE (cont.)The results demonstrate linearity as well over the specified range.Correlation coefficient (RSQ)0.99981 Slope11808.3Y -Interceptresponse at 100%* 100 (%) 0.3%ACCURACYACCURACY OF STANDARD INJECTIONSFive (5) replicate injections of the working standard solution at concentration of 0.4242mg/mL, as described in method SI-IAG-206-02 were made.INJECTIONNO.PEAK AREA%ACCURACYI 539299.7II 540599.9III 540499.9IV 5406100.0V 5407100.0Average 5402.899.9%SD 6.10.1RSD, (%)0.10.1The percent deviation from the true value wasdetermined from the linear regression lineHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::ACCURACY OF THE DRUG PRODUCTAdmixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl were prepared at the same proportion as in a capsule (70%-180% of the nominal concentration).Three preparations were made for each concentration and the recovery was calculated.Conc.(%)Placebo Wt.(mg)Fluoxetine HCl Wt.(mg)Peak Area%Accuracy Average (%)70%7079.477.843465102.27079.687.873427100.77079.618.013465100.0101.0100%10079.6211.25476397.910080.8011.42491799.610079.6011.42485498.398.6130%13079.7214.90640599.413080.3114.75632899.213081.3314.766402100.399.618079.9920.10863699.318079.3820.45879499.418080.0820.32874899.599.4Placebo, Batch Lot IAG-21-001HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION OF FLUOXETINE HClAT LOW CONCENTRATIONLINEARITY AT LOW CONCENTRATIONSStandard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of the working concentration required by the method SI-IAG-206-02. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over this range.ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATIONThe peak areas of the standard solution at the working concentration were measured and the percent deviation from the true value, as determined from the linear regression was calculated.SAMPLECONC.µg/100mLAREA FOUND%ACCURACYI 470.56258499.7II 470.56359098.1III 470.561585101.3IV 470.561940100.7V 470.56252599.8VI 470.56271599.5(%) AverageSlope = 132.7395299.9SD Y-Intercept = -65.872371.1(%) RSD1.1HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSystem RepeatabilitySix replicate injections of standard solution at 0.02% and 0.05% of working concentration as described in method SI-IAG-206-02 were made and the relative standard deviation was calculated.SAMPLE FLUOXETINE HCl AREA0.02%0.05%I10173623II11503731III10103475IV10623390V10393315VI10953235Average10623462RSD, (%) 5.0 5.4Quantitation Limit - QLThe quantitation limit ( QL) was established by determining the minimum level at which the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02% of the working standard concentration with resulting RSD (for six injections) of 5.0%. Detection Limit - DLThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected. The detection limit of Fluoxetine HCl is about 0.01% of the working standard concentration.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION FOR META-FLUOXETINE HCl(EVALUATING POSSIBLE IMPURITIES)Meta-Fluoxetine HCl linearity at 0.05% - 1.0%Relative Response Factor (F)Relative response factor for Meta-Fluoxetine HCl was determined as slope of Fluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs (analysed at the same time).F =132.7395274.859534= 1.8Detection Limit (DL) for Fluoxetine HClThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected.Detection limit for Meta Fluoxetine HCl is about 0.02%.Quantitation Limit (QL) for Meta-Fluoxetine HClThe QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-Fluoxetine HCl can be quantified with acceptable accuracy and precision.Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HCl solution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into the HPLC and the recovery was calculated.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES].Approx.Conc.(%)Known Conc.(µg/100ml)Area in SpikedSampleFound Conc.(µg/100mL)Recovery (%)0.0521.783326125.735118.10.0521.783326825.821118.50.0521.783292021.55799.00.0521.783324125.490117.00.0521.783287220.96996.30.0521.783328526.030119.5(%) AVERAGE111.4SD The recovery result of 6 samples is between 80%-120%.10.7(%) RSDQL for Meta Fluoxetine HCl is 0.05%.9.6Accuracy for Meta Fluoxetine HClDetermination of Accuracy for Meta-Fluoxetine HCl impurity was assessed using triplicate samples (of the drug product) spiked with known quantities of Meta Fluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and 120% of the specified limit - 0.05%).The results are within specifications:For 0.4% and 0.5% recovery of 85% -115%For 0.6% recovery of 90%-110%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES]Approx.Conc.(%)Known Conc.(µg/100mL)Area in spikedSample Found Conc.(µg/100mL)Recovery (%)[0.4%]0.4174.2614283182.66104.820.4174.2614606187.11107.370.4174.2614351183.59105.36[0.5%]0.5217.8317344224.85103.220.5217.8316713216.1599.230.5217.8317341224.81103.20[0.6%]0.6261.3918367238.9591.420.6261.3920606269.81103.220.6261.3920237264.73101.28RECOVERY DATA DETERMINED IN SPIKED SAMPLESHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::REPEATABILITYMethod Repeatability - Meta Fluoxetine HClThe full method (as described in SI-IAG-206-02) was carried out on the finished drug product representing lot number IAG-21-001-(1). The HPLC method repeated serially, six times and the relative standard deviation (RSD) was calculated.IAG-21-001 20mg CAPSULES - FLUOXETINESample% Meta Fluoxetine % Meta-Fluoxetine 1 in Spiked Solution10.0260.09520.0270.08630.0320.07740.0300.07450.0240.09060.0280.063AVERAGE (%)0.0280.081SD 0.0030.012RSD, (%)10.314.51NOTE :All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta Fluoxetine HCl were injected.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::Intermediate Precision - Meta-Fluoxetine HClThe full method as described in SI-IAG-206-02 was applied on the finished product IAG-21-001-(1) .It was repeated six times, with a different analyst on a different day using a different HPLC instrument.The difference between the average results obtained by the method repeatability and the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HCl as is and 28.5% for spiked solution).IAG-21-001 20mg - CAPSULES FLUOXETINESample N o:Percentage Meta-fluoxetine% Meta-fluoxetine 1 in spiked solution10.0260.06920.0270.05730.0120.06140.0210.05850.0360.05560.0270.079(%) AVERAGE0.0250.063SD 0.0080.009(%) RSD31.514.51NOTE:All results obtained were well below the QL (0.05%) thus spiked samples slightly greater than 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solution was 14.5%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSPECIFICITY - STABILITY INDICATING EVALUATIONDemonstration of the Stability Indicating parameters of the HPLC assay method [SI-IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array detector was incorporated utilizing a commercial chromatography software managing system2, and applied to analyze a range of stressed samples of the finished drug product.GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):Purity Angle-is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold-is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.Match Angle-is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold-is the sum of the match noise angle3 and match solvent angle4.3Noise Angle-is a measure of spectral non-homogeneity caused by system noise.4Solvent Angle-is a measure of spectral non-homogeneity caused by solvent composition.OVERVIEWT he assay of the main peak in each stressed solution is calculated according to the assay method SI-IAG-206-02, against the Standard Solution, injected on the same day.I f the Purity Angle is smaller than the Purity Threshold and the Match Angle is smaller than the Match Threshold, no significant differences between spectra can be detected. As a result no spectroscopic evidence for co-elution is evident and the peak is considered to be pure.T he stressed condition study indicated that the Fluoxetine peak is free from any appreciable degradation interference under the stressed conditions tested. Observed degradation products peaks were well separated from the main peak.1® PDA-996 Waters™ ; 2[Millennium 2010]ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFORCED DEGRADATION OF FINISHED PRODUCT & STANDARD 1.UNSTRESSED SAMPLE1.1.Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 98.5%.SAMPLE - UNSTRESSEDFluoxetine:Purity Angle:0.075Match Angle:0.407Purity Threshold:0.142Match Threshold:0.4251.2.Standard solution was prepared as stated in method SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 100.0%.Fluoxetine:Purity Angle:0.078Match Angle:0.379Purity Threshold:0.146Match Threshold:0.4272.ACID HYDROLYSIS2.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system after filtration.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.SAMPLE- ACID HYDROLYSISFluoxetine peak:Purity Angle:0.055Match Angle:0.143Purity Threshold:0.096Match Threshold:0.3712.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc. HCl were added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSTANDARD - ACID HYDROLYSISFluoxetine peak:Purity Angle:0.060Match Angle:0.060Purity Threshold:0.099Match Threshold:0.3713.BASE HYDROLYSIS3.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.SAMPLE - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.063Match Angle:0.065Purity Threshold:0.099Match Threshold:0.3623.2.Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease - 99.5%.STANDARD - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.081Match Angle:0.096Purity Threshold:0.103Match Threshold:0.3634.OXIDATION4.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent added and the solution sonicated for 10 minutes.1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then made up to volume with Diluent, filtered and injected into HPLC system.Fluoxetine peak intensity decreased to 95.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSAMPLE - OXIDATIONFluoxetine peak:Purity Angle:0.090Match Angle:0.400Purity Threshold:0.154Match Threshold:0.4294.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of 30% H2O2 were added to this solution which was standing for 5 hours, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity decreased to 95.8%.STANDARD - OXIDATIONFluoxetine peak:Purity Angle:0.083Match Angle:0.416Purity Threshold:0.153Match Threshold:0.4295.SUNLIGHT5.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST was set to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%. The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak was observed at RRT of 1.5 (2.7%).The total percent of Fluoxetine peak with the degradation peak is about 93.9%.SAMPLE - SUNLIGHTFluoxetine peak:Purity Angle:0.093Match Angle:0.583Purity Threshold:0.148Match Threshold:0.825 ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSUNLIGHT (Cont.)5.2.Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACT was 42°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of 99.5%.The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak were observed at RRT of 1.5 (2.3).The total percent of Fluoxetine peak with the degradation peak is about 97.5%. STANDARD - SUNLIGHTFluoxetine peak:Purity Angle:0.067Match Angle:0.389Purity Threshold:0.134Match Threshold:0.8196.HEAT OF SOLUTION6.1.Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution was sonicated for 10 minutes and made up to volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and injected into the HPLC system.Fluoxetine peak was decreased to 93.3%.SAMPLE - HEAT OF SOLUTION [105o C]Fluoxetine peak:Purity Angle:0.062Match Angle:0.460Purity Threshold:0.131Match Threshold:0.8186.2.Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL of the working solution was transferred into a suitable crucible, placed in an oven at 105°C for 2 hours, cooled to ambient temperature and injected into the HPLC system.Fluoxetine peak intensity did not decrease - 100.5%.ED. N0: 04Effective Date:APPROVED::。
抗可提取性核抗原(ENA)抗体(6种)定性 化学发光蛋白 …
【包装规格】 48 人份/盒
【临床意义】 抗可提取性核抗原（ENA）抗体（6 种）定性检测试剂盒(化学发光蛋白芯片法)定性检 测血清样本中的 6 种可提取抗核抗体，包括 SSA/Ro、SSB/La、Jo-1、RNP、Sm、Scl-70。 它们的临床意义如下： （1）抗 SSA／Ro 抗体：SSA／Ro 是小分子细胞浆核糖核蛋白（scRNPs） ，是蛋白 和小分子核糖核酸形成的复合物。 抗原是含有 Y1－Y5 RNA 的蛋白质， 其分子量有 52KD 及 60KD。52KD 的多肽条带与干燥综合征（SS）相关，而 60KD 的多肽条带则更多存 在于 SLE 患者中。抗 SSA 抗体主要见于原发性干燥综合征，阳性率高达 60％～75％。 此外， 抗 SSA 抗体常与亚急性皮肤性红斑狼疮、 抗核抗体阴性狼疮、 新生儿狼疮等相关。 （2）抗 SSB/La/Ha 抗体：SSB 抗原是 RNA 多聚酶转录中的小 RNA 磷酸蛋白质。 其分子量为 48KD、47KD、45KD，其中 48KD 更具特异性。抗 SSB 抗体较抗 SSA 抗体 诊断干燥综合征更特异，是干燥综合征血清特异性抗体。原发性干燥综合征阳性率达 40％左右。其他自身免疫性疾病中如有抗 SSB 抗体，常伴有继发性干燥综合征。 （3）抗 Scl-70 抗体：天然 Scl-70 抗原是分子量为 100KD 的 DNA 拓朴异构酶 I 的 降解产物，因其主要见于硬皮病，且其相应抗原分子量为 70KD，故取名为抗 Scl-70 抗 体。系统性硬化症中阳性率达 20%～59%，重症弥漫性 PSS（SSc）中抗 Scl-70 抗体阳 性率高达 75％。
湖州数康生物科技有限公司
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（4）抗 Jo-1 抗体：Jo-1 抗原是组氨酰-tRNA 合成酶在胞浆中以小分子核糖核蛋白 （scRNPs）形式出现，分子量为 50KD。抗 Jo-1 抗体对多发性肌炎/皮肌炎（PM/DM） 的诊断具有较强的特异性，阳性率为 25％-35%。 （5）抗 RNP 抗体：临床上应用较多的是 U1RNP 抗体，U1snRNP 由 U1RNP 和 9 种不同的蛋白质组成，所作用的抗原是 U1 小分子细胞核核糖核蛋白（U1snRNP） ，所以 又称抗 U1RNP 抗体。混合性结缔组织病(MCTD)的抗 RNP 阳性率>95%。抗体滴度与疾 病活动相关。抗 RNP 抗体在 SLE 中的阳性率为 40%左右，但几乎总伴有抗 Sm 抗体。 （6）抗 Sm 抗体： Sm 抗原是 U 族小分子细胞核核糖核蛋白（UsnRNP） 。Sm 抗体 和 SnRNP 是同一分子复合物中的不同抗原位点，故抗 Sm 抗体很少单独出现，它常于 U1RNP 抗体相伴，在 SLE 中阳性率为 30.2%，为 SLE 的标记抗体。
美国贝克曼库尔特流式细胞分析仪
美国贝克曼库尔特流式细胞分析仪（Beckman coulter cell）产品型号：Cell Lab Quanta SC当前价格：0.00元产品数量：0新旧程度：全新有效期至：0000-00-00所在地：产品简介：仪器简介：T细胞亚群检测的CD45/CD4/CD8/CD3、CD45/CD56/CD19/CD3；阵发性血红蛋白尿（PNH）检测的CD55、CD59；血小板无力症（GT）检测的CD41、CD61等等详细信息仪器简介：T细胞亚群检测的CD45/CD4/CD8/CD3、CD45/CD56/CD19/CD3；阵发性血红蛋白尿（PNH）检测的CD55、CD59；血小板无力症（GT）检测的CD41、CD61等等。
但对于白血病/淋巴瘤免疫分型，国际上迄今为止也没有统一的抗体组合。
在2000年国际细胞分析学会（ISAC）大会上，临床血细胞计数协会组织了一次国际专家会议，以期对检测血液淋巴系统肿瘤所需最少、最有效的单抗数达成共识。
75%与会者一致认为，对于慢性淋巴系统增殖性疾病（CLD）有9种单抗：CD5,CD19,κ,λ,CD3,CD20,CD23,CD10,CD45对初诊来说是最基本的。
淋巴瘤和CLD相似，需要至少12-16种单抗。
对于急性白血病（AL），75%的与会者认为大约13-15种单抗是最基本的：CD10,CD19,CD79a,CD13,CD33,CD34,CD45,CD2,MPO，CD7,CD14,CD3,HLA-DR等，对初步鉴别白血病系列是必需的。
其他一些（CD16,CD56,CDw65,TdT,cyCD3）可能对某些病例有用。
几乎所有的投票者都认为，要对急性白血病完善分类所需单抗的恰当数量平均为20-24种。
但这些抗体之间组合也是一大难题，目前也无统一规定（如表二）。
大会多数发言者(11/13)指出，对已确诊病人的监护和分期来说，仅需较少单抗。
抗体的质量控制是实验的关键环节。
抗体的质量包括其特异性、灵敏度、精密度。
大鼠硫酸乙酰肝素(HS)酶联免疫吸附测定试剂盒 说明书
Uscn Life Science Inc. Wuhan网址: 电话: +86 27 84259552传真: +86 27 84259551E-mail:***************大鼠硫酸乙酰肝素(HS)酶联免疫吸附测定试剂盒使用说明书产品编号：E0161Ra规格：96T本试剂盒仅供体外研究使用，不用于临床诊断!预期应用本酶联免疫吸附测定试剂盒运用双抗体夹心ELISA法定量测定大鼠血清、血浆、组织匀浆或其它相关生物液体中HS含量。
本试剂盒已提供的试剂试剂名称数量96孔板(预包被) 1标准品(冻干) 2标准品稀释液 1 × 20ml检测溶液A 1 × 120μl检测溶液B 1 × 120μl检测稀释液A(2 x) 1 × 6ml检测稀释液B(2 x) 1 × 6mlTMB底物 1 × 9ml终止液 1 × 6ml浓洗涤液(30 x) 1 × 20ml96孔板覆膜 4使用说明书 1本试剂盒未提供但需自备的设备及试剂1、450±10nm滤光片的酶标仪（建议仪器使用前提前预热）2、单道和多道微量加液器及吸头3、稀释样品的EP管4、蒸馏水或去离子水5、吸水纸6、盛放洗液的容器试剂盒的储存及有效期所有试剂均按试剂瓶标签上所示保存。
请注意，收到试剂盒后请尽快将标准品、检测溶液A、检测溶液B以及96孔板保存于-20。
开封后的酶标板要密封加干燥剂后保存于-20，避免潮湿。
有效期为6个月。
实验原理将HS抗体包被于96孔微孔板中，制成固相载体，向微孔中依次加入标准品和标本，其中的HS与连接于固相载体上的抗体结合，洗板之后加入生物素化的HS抗体，将未结合的生物素化抗体洗净后，加入HRP标记的亲和素，再次彻底洗涤后加入底物(TMB)显色。
TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的HS呈正相关。
IPI549-SDS-MedChemExpress
Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:Oct.-12-2018Print Date:Oct.-12-20181. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :IPI549Catalog No. :HY-100716CAS No. :1693758-51-81.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureGHS Classification in accordance with 29 CFR 1910 (OSHA HCS)Acute toxicity, Oral (Category 4), H302Acute aquatic toxicity (Category 1), H400Chronic aquatic toxicity (Category 1), H4132.2 GHS Label elements, including precautionary statementsPictogramSignal word No data availableHazard statement(s)H302 Harmful if swallowed.H413 Very toxic to aquatic life with long lasting effects.Precautionary statement(s)P264 Wash skin thoroughly after handling.P270 Do not eat, drink or smoke when using this product.P273 Avoid release to the environment.P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor ⁄physician if you feel unwell.P333 Rinse mouth.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:IPI-549;IPI 549Formula:C30H24N8O2Molecular Weight:528.56CAS No. :1693758-51-84. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature:Powder-20°C 3 years4°C 2 yearsIn solvent-80°C 6 months-20°C 1 monthShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance White to yellow (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. STABILITY AND REACTIVITY10.1 ReactivityNo data available.10.2 Chemical stabilityStable under recommended storage conditions.10.3 Possibility of hazardous reactionsNo data available.10.4 Conditions to avoidNo data available.10.5 Incompatible materialsStrong acids/alkalis, strong oxidising/reducing agents.10.6 Hazardous decomposition productsUnder fire conditions, may decompose and emit toxic fumes.Other decomposition products - no data available.11.TOXICOLOGICAL INFORMATION11.1 Information on toxicological effectsAcute toxicityClassified based on available data. For more details, see section 2Skin corrosion/irritationClassified based on available data. For more details, see section 2Serious eye damage/irritationClassified based on available data. For more details, see section 2Respiratory or skin sensitizationClassified based on available data. For more details, see section 2Germ cell mutagenicityClassified based on available data. For more details, see section 2CarcinogenicityIARC: No component of this product present at a level equal to or greater than 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.ACGIH: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by ACGIH.NTP: No component of this product present at a level equal to or greater than 0.1% is identified as a anticipated or confirmed carcinogen by NTP.OSHA: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by OSHA.Reproductive toxicityClassified based on available data. For more details, see section 2Specific target organ toxicity - single exposureClassified based on available data. For more details, see section 2Specific target organ toxicity - repeated exposureClassified based on available data. For more details, see section 2Aspiration hazardClassified based on available data. For more details, see section 212. ECOLOGICAL INFORMATION12.1 ToxicityNo data available.12.2 Persistence and degradabilityNo data available.12.3 Bioaccumlative potentialNo data available.12.4 Mobility in soilNo data available.12.5 Results of PBT and vPvB assessmentPBT/vPvB assessment unavailable as chemical safety assessment not required or not conducted.12.6 Other adverse effectsNo data available.13. DISPOSAL CONSIDERATIONS13.1 Waste treatment methodsProductDispose substance in accordance with prevailing country, federal, state and local regulations.Contaminated packagingConduct recycling or disposal in accordance with prevailing country, federal, state and local regulations.14. TRANSPORT INFORMATIONDOT (US)This substance is considered to be non-hazardous for transport.IMDGUN number: 3077Class: 12Packing group: IIIEMS-No: F-A, S-FProper shipping name: ENVIRONMENTALLY HAZARDOUS SUBSTANCE, SOLID, N.O.S.Marine pollutant: Marine pollutantIATAUN number: 3077Class: 12Packing group: IIIProper shipping name: Environmentally hazardous substance, solid, n.o.s.15. REGULATORY INFORMATIONSARA 302 Components:No chemicals in this material are subject to the reporting requirements of SARA Title III, Section 302.SARA 316 Components:This material does not contain any chemical components with known CAS numbers that exceed thethres33&33U_HKSCS33&MingLiU_HKSCS3333333333316. OTHER INFORMATIONCopyright 2018 MedChemExpress. The above information is correct to the best of our present knowledge but does not purport to be all inclusive and should be used only as a guide. The product is for research use only and for experienced personnel. It must only be handled by suitably qualified experienced scientists in appropriately equipped and authorized facilities. The burden of safe use of this material rests entirely with the user. MedChemExpress disclaims all liability for any damage resulting from handling or from contact with this product.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。
Adenosine Assay Kit 产品说明书
Product ManualAdenosine Assay KitCatalog NumberMET-5090 100 assays FOR RESEARCH USE ONLYNot for use in diagnostic proceduresIntroductionAdenosine is a purine nucleoside containing an adenine moiety attached to a ribose sugar molecule (ribofuranose) through a β-N9-glycosidic bond. Derivatives of adenosine perform an important role in energy transfer reactions (as adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) as well as signal transduction (as cyclic adenosine monophosphate (cAMP)). Additionally, adenosine is a neuromodulator and is thought to promote sleep and suppress arousal. Adenosine also regulates blood flow to multiple organs through vasodilation. Adenosine is a byproduct of the enzymatic conversion of S-Adenosylhomocysteine (SAH) to homocysteine by Adenosylhomocystinease (AHCY).Adenosine causes a temporary block of the atrioventricular (AV) node in the heart, while also relaxing smooth muscle found inside the artery walls. Dilation of the "normal" segments of arteries allows physicians to use adenosine to test for blockages in the coronary arteries, by exaggerating the difference between the normal and abnormal segments. In people suspected of having a supraventricular tachycardia (SVT), adenosine can be used to help identify the problem. Certain SVTs can be successfully stopped with adenosine. In addition, atrial tachycardia can sometimes be terminated with adenosine. Finally, adenosine is used in combination with thallous (thallium) chloride TI 201 or Tc99m myocardial perfusion scintigraphy (nuclear stress test for heart attack risk) in people who are unable to undergo sufficient stress testing with exercise.Ce ll Biolabs’ Adenosine Assay Kit is a simple fluorometric assay that measures the amount of total adenosine present in biological samples in a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays*, including blanks, adenosine standards, and unknown samples. Sample adenosine concentrations are determined by comparison with a known adenosine standard. The kit has a detection sensitivity limit of 1.56 µM adenosine.*Note: Each sample replicate requires 2 assays, one treated with adenosine deaminase (+ADA) and one without (-ADA). Adenosine is calculated from the difference in RFU readings from the 2 wells. Assay PrincipleCell Biolabs’ Adenosine Assay Kit measures total adenosine within biological samples. Adenosine is converted into inosine by adenosine deaminase (ADA). Then inosine is converted into hypoxanthine by purine nucleoside phosphorylase (PNP). Finally, hypoxanthine is converted to xanthine and hydrogen peroxide by xanthine oxidase (XO). The resulting hydrogen peroxide is then detected with a highly specific fluorometric probe. Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide, which bind in a 1:1 ratio. Samples are compared to a known concentration of adenosine standard within the 96-well microtiter plate format. Samples and standards are incubated for 15 minutes and then read with a standard 96-well fluorometric plate reader (Figure 1).Figure 1.Adenosine Assay Principle.Related Products1.MET-5092: Inosine Assay Kit2.MET-5151: S-Adenosylhomocysteine (SAH) ELISA Kit3.MET-5152: S-Adenosylmethionine (SAM) ELISA KitKit Components1.Adenosine Standard (Part No. 50901C): One 50 µL tube at 10 mM.2.10X Assay Buffer (Part No. 268002): One 25 mL bottle of 500 mM sodium phosphate pH 7.4.3.Fluorometric Probe (Part No. 50231C): One 50 µL tube in DMSO.4.HRP (Part No. 234402-T): One 10 μL t ube of a 100 U/mL solution in glycerol.5.Adenosine Deaminase (Part No. 50902C): One 10 µL tube at 1000 U/mL.Note: One unit is defined as the amount of enzyme that will deaminate 1.0 μmole of adenosine to inosine per min. at pH 7.5 at 25°C.6.Purine Nucleoside Phosphorylase (Part No. 50903D): One 500 µL tube at 18.9 U/mL.Note: One unit is defined as the amount of enzyme that will cause the phosphorolysis of 1.0 μmole of inosine to hypoxanthine and ribose 1-phosphate per min at pH 7.4 at 25°C.7.Xanthine Oxidase (Part No. 50904D): one 100 µL tube at 2.5 U/mL.Note: One unit is defined as the amount of enzyme that will convert 1.0 μmol e of xanthine to uric acid per min at pH 7.5 at 25°C. About 50% of the activity is obtained with hypoxanthine as substrate.Materials Not Supplied1.Phosphate Buffered Saline (PBS)2.10 μL to 1000 μL adjustable single channel micropipettes with disposable ti ps3.50 μL to 300 μL adjustable multichannel micropipette with disposable tips4.Standard 96-well fluorescence black microtiter plate and/or black cell culture microplate5.Multichannel micropipette reservoir6.Fluorescence microplate reader capable of reading excitation in the 530-570 nm range and emissionin the 590-600 nm range.StorageUpon receipt, store the 10X Assay Buffer at room temperature and store the rest of the kit at -20ºC. The Fluorometric Probe is light sensitive and must be stored accordingly. Avoid multiple freeze/thaw cycles.Note: After thawing any of the three enzymes for the first time, make smaller aliquots and store at-20°C.Preparation of Reagents•1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution.Stir or vortex to homogeneity. Store at room temperature.•Reaction Mix: Prepare a Reaction Mix by diluting the Fluorometric Probe 1:100, HRP 1:500, Adenosine Deaminase 1:500, Purine Nucleoside Phosphorylase 1:10, and Xanthine Oxidase 1:50 in 1X Assay Buffer. For example, add 10 μL Fluorometric Probe stock solution, 2 μL HRP stock solution, 2 µL of Adenosine Deaminase, 100 µL of Purine Nucleoside Phosphorylase, and 20 μL of Xanthine Oxidase to 866 µL of 1X Assay Buffer for a total of 1 mL. This Reaction Mix volume is enough for 20 assays. The Reaction Mix is stable for 1 day at 4ºC.Note: Prepare only enough for immediate use by scaling the above example proportionally. •Control Mix: Prepare a Reaction Mix (without adenosine deaminase) by diluting the Fluorometric Probe 1:100, HRP 1:500, Purine Nucleoside Phosphorylase 1:10, and Xanthine Oxidase 1:50 in 1X Assay Buffer. For example, add 10 μL Fluorometric Probe stock solution, 2 μL HRP stocksolution, 100 µL of Purine Nucleoside Phosphorylase, and 20 μL of Xanthine Oxidase to 868 µL of 1X Assay Buffer for a total of 1 mL. This Control Mix volume is enough for 20 assays. TheControl Mix is stable for 1 day at 4ºC.Note: Prepare only enough for immediate use by scaling the above example proportionally.Preparation of Samples• Cell culture supernatants: Cell culture media containing adenosine, inosine, xanthine, and hypoxanthine should be avoided. To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The cell conditioned media may be assayed directly or diluted as necessary in PBS.Note: Maintain pH between 7 and 8 for optimal working conditions as the Fluorometric Probe is unstable at high pH (>8.5).• Tissue lysates: Sonicate or homogenize tissue sample in PBS and centrifuge at 10,000 x g for 10 minutes at 4°C. The supernatant may be assayed directly or diluted as necessary in PBS.• Cell lysates: Resuspend cells at 1-2 x 106 cells/mL in PBS. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates may be assayed undiluted or diluted as necessary in PBS.• Serum, plasma or urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant may be assayed directly or diluted as necessary in PBS.Notes:• All samples should be assayed immediately or stored at -80°C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.• Samples with NADH concentrati ons above 10 μM and glu tathione concentrations above 50 μM will oxidize the Fluorometric Probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL (Votyakova and Reynolds, Ref. 2).• Avoid samples containing DTT or β-mercaptoethanol since the Fluorometric Probe is not stable in the presence of thiols (above 10 μM).Preparation of Standard CurvePrepare fresh Adenosine standards according to Table 1 below.Table 1. Preparation of Adenosine Standards.Assay Protocol1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate. Standard Tubes 10 mM Adenosine Solution(µL)PBS (µL) Adenosine (µM) 1 5495 100 2 250 of Tube #1250 50 3 250 of Tube #2250 25 4 250 of Tube #3250 12.5 5 250 of Tube #4250 6.25 6 250 of Tube #5250 3.13 7 250 of Tube #6250 1.56 8 0250 0Note: Each sample replicate requires two paired wells, one to be treated with Adenosine Deaminase (+ADA) and one without the enzyme (-ADA) to measure endogenous background.2.Add 50 μL of each standard into wells of a black microtiter plate suitable for a f luorescence platereader.3.Add 50 μL of each unknown sample to each of two separate wells.4.Add 50 μL of Reaction Mix to all standard wells and one half of the paired sample wells.5.Add 50 μL of Control Mix to the remaining paired sample wells.6.Mix the well contents thoroughly and incubate for 15 minutes at room temperature protected fromlight.Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the reaction kinetics.7.Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nmrange and for emission in the 590-600 nm range.Example of ResultsThe following figure demonstrates typical Adenosine Assay Kit results. One should use the data below for reference only. This data should not be used to interpret or calculate actual sample results.Figure 2: Adenosine Standard Curve.Calculation of Results1.Determine the average Relative Fluorescence Unit (RFU) values for each sample, control, andstandard.2.Subtract the average zero standard value from itself and all standard values.3.Graph the standard curve (see Figure 2).4.Subtract the sample well values without Adenosine Deaminase (-ADA) from the sample well valuescontaining Adenosine Deaminase (+ADA) to obtain the difference. The fluorescence difference is due to the Adenosine Deaminase activity.Net RFU = (RFU+ADA)- (RFU-ADA)5. Compare the net RFU of each sample to the standard curve to determine and extrapolate thequantity of Adenosine present in the sample. Only use values within the range of the standard curve.References1.Sato, A (A2005). Am. J. of Physiol.Heart Circ. Physiol.288: H1633–402.Votyakova TV, and Reynolds IJ (2001) Neurochem. 79:266.3.Costa, F and Biaggioni, I (1998). Hypertension. 31: 1061–44.Morgan, JM; McCormack, DG; Griffiths, MJ; Morgan, CJ; Barnes, PJ and Evans, TW (1991).Circulation. 84: 1145–9.5.Mitchell J and Lazarenko G (2008). CJEM. 10: 572–3.6.O'Keefe, JH; Bateman, TM; Silverstri, R and Barnhart C. (1992). Am Heart J.124: 614–21. Recent Product Citations1.Ndzie Noah, M.L. et al. (2023). Estrogen downregulates CD73/adenosine axis hyperactivity viaadaptive modulation PI3K/Akt signaling to prevent myocarditis and arrhythmias during chronic catecholamines stress. Cell Commun Signal. 21(1):41. doi: 10.1186/s12964-023-01052-0.2.Fu, Z. et al. (2023). Proteolytic regulation of CD73 by TRIM21 orchestrates tumor immunogenicity.Sci Adv. 9(1):eadd6626. doi: 10.1126/sciadv.add6626.3.Pepponi, R. et al. (2022). Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof-of-Concept Study. Int J Mol Sci. 23(7):3456. doi: 10.3390/ijms23073456.4.Kim, G.T. et al. (2022). PLAG co-treatment increases the anticancer effect of Adriamycin andcyclophosphamide in a triple-negative breast cancer xenograft mouse model. Biochem Biophys Res Commun. 619:110-116. doi: 10.1016/j.bbrc.2022.06.051.5.Tsai, C.H. et al. (2022). Carbohydrate metabolism is a determinant for the host specificity ofbaculovirus infections. iScience. doi: 10.1016/j.isci.2021.103648.6.Xue, G. et al. (2021). Elimination of acquired resistance to PD-1 blockade via the concurrentdepletion of tumour cells and immunosuppressive cells. Nat Biomed Eng. 5(11):1306-1319. doi:10.1038/s41551-021-00799-6.7.Murphy, D.A. et al. (2021). Reversing Hypoxia with PLGA-Encapsulated Manganese DioxideNanoparticles Improves Natural Killer Cell Response to Tumor Spheroids. Mol Pharm. doi:10.1021/acs.molpharmaceut.1c00085.8.Badimon, A. et al. (2020). Negative feedback control of neuronal activity by microglia. Nature. doi:10.1038/s41586-020-2777-8.9.Huang, L. et al. (2020). Inhibition of A2B Adenosine Receptor Attenuates Intestinal Injury in a RatModel of Necrotizing Enterocolitis. Mediators Inflamm. doi: 10.1155/2020/1562973.10.Basu, M. et al. (2020). Increased host ATP efflux and its conversion to extracellular adenosine iscrucial for establishing Leishmania infection. J Cell Sci. pii: jcs.239939. doi: 10.1242/jcs.239939.11.Duan, L. et al. (2020). Late Protective Effect of Netrin-1 in the Murine AcetaminophenHepatotoxicity Model. Toxicol Sci. pii: kfaa041. doi: 10.1093/toxsci/kfaa041.12.Chang, Y. et al. (2020). Snellenius manilae bracovirus suppresses the host immune system byregulating extracellular adenosine levels in Spodoptera litura. Sci Rep. 10(1):2096. doi:10.1038/s41598-020-58375-y.13.Ali, R.A. et al. (2019). Adenosine receptor agonism protects against NETosis and thrombosis inantiphospholipid syndrome. Nat Commun. 10(1):1916. doi: 10.1038/s41467-019-09801-x. WarrantyThese products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF F ITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products. In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with the products.Contact InformationCell Biolabs, Inc.7758 Arjons DriveSan Diego, CA 92126Worldwide: +1 858 271-6500USA Toll-Free: 1-888-CBL-0505E-mail: ********************©2017-2023: Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without permissions in writing.。
Incucyte
Product Information Presentation, Storage and StabilityThe Incucyte® Fabfluor-pH Antibody Labeling Reagents for antibody internalization are supplied as lyophilized solids in sufficient quantity to label 50 μg of test antibody, when used at the suggested molar ratio (1:3 of test antibody to labeling Fab). The lyophilized solid can be stored at 2-8° C for one year. Once re-hydrated, any unused reagent should be aliquoted and stored at -80° C for up to one year. Avoid repeated freeze-thaw cycles.Incucyte® Fabfluor-pH Antibody Labeling ReagentsFor Antibody Internalization AssaysAntibody Labeling Reagent Rehydrated: -80° C *Excitation and Emission maxima were determined at a pH of 4.5.Fabfluor_quick_guideBackgroundIncucyte ® Fabfluor-pH Antibody Labeling Reagents are designed for quick, easy labeling of Fc-containing test antibodies with a Fab fragment-conjugated pH-sensitive fluorophore. The pH-sensitive dye based system exploits the acidic environment of the lysosomes to quantify in-ternalization of the labeled antibody. As Fabfluor labeled antibodies reside in the neutral extracellular solution (pH 7.4), they interact with cell surface specific antigens and are internalized. Once in the lysosomes, they enter an acidic environment (pH 4.5–5.5) and a substantial in-crease in fluorescence is observed. In the absence of ex-pression of the specific antigen, no internalization occurs and the fluorescence intensity of the labeled antibodies remains low. With the Incucyte ® integrated analysis soft-ware, background fluorescence is minimized. These reagents have been validated for use with a number of different antibodies in a range of cell types. The Incucyte ® Live-Cell Analysis System enables real-time, kinetic eval -uation of antibody internalization.Recommended UseWe recommend that the Incucyte ® Fabfluor-pH Antibody Labeling Reagents are prepared at a stock concentration of 0.5 mg/mL by the addition of 100 μL of sterile water and triturated (centrifuge if solution not clear). The reagent may then be diluted directly into the labeling mixture with test antibody. Do NOT sonicate the solution.Additional InformationThe Fab antibody was purified from antisera by a combination of papain digestion and immunoaffinity chromatography using antigens coupled to agarose beads. Fc fragments and whole IgG molecules have been removed.Human Red (Cat. No. 4722) or Human Orange (Cat. No. 4812)—Based on immunoelectrophoresis and/ or ELISA, the antibody reacts with the Fc portion of human IgG heavy chain but not the Fab portion of human IgG. No antibody was detected against human IgM, IgA or against non-immunoglobulin serum proteins. The anti-body may cross-react with other immunoglobulins from other species.Mouse IgG1 (Cat. No. 4723), IgG2a (Cat. No. 4750) or IgG2b (Cat. No. 4751)—Based on antigen-binding assay and/or ELISA, the antibody reacts with the Fc portion of mouse IgG, IgG2a or IgG2b, respectively, but not the Fab portion of mouse immunoglobulins. No antibody was detected against mouse IgM or against non–immunoglobulin serum proteins. The antibody may cross-react with other mouse IgG subclasses or with immunoglobulins from other species.Rat (Cat. No. 4737)—Based on immunoelectrophoresis and/or ELISA, the antibody reacts with the Fc portion of rat IgG heavy chain but not the Fab portion of rat IgG. No antibody was detected against rat IgM, IgA or against non-immunoglobulin serum proteins. The antibody may cross-react with other immunoglobulins from other species.A.B.C.D.R e d O b j e c t A r e a (x 105 μm 2 p e r w e l l )Time (hours)A U C x 106 (0–12 h )log [α–CD71] (g/mL)Example DataFigure 1: Concentration-dependent increase in antibody internalization of Incucyte ® Fabfluor labeled-α-CD71 in HT1080 cells. α-CD71 and mouse IgG1 isotype control were labeled with Incucyte ® Mouse IgG1 Fabfluor-pH Red Antibody Labeling Reagent. HT1080 cells were treated with either Fabfluor-α-CD71 or Fabfluor-IgG1 (4 μg/mL); HD phase and red fluorescence images were captured every 30 minutes over 12 hours using a 10X magnification. (A) Images of cells treated with Fabfluor-α-CD71 display red fluorescence in the cytoplasm (images shown at 6 h). (B) Cells treated with labeled isotype control display no cellular fluorescence. (C) Time-course of Fabfluor-α-CD71 internalization with increasing concentrations of Fabfluor-α-CD71 (progressively darker symbols). Internalization has been quantified as the red object area for each time-point. (D) Concentration response curve to Fabfluor-α-CD71. Area under the curve (AUC) values have been determined from the time-course shown in panel C (0-12 hours) and are presented as the mean ± SEM, n=3 wells.CD71-FabfluorIgG-FabfluorProtocols and ProceduresMaterialsIncucyte® Fabfluor-pH Antibody Labeling ReagentTest antibody of interest containing human, mouse, or rat IgG Fc region (at known concentration)Target cells of interestTarget cell growth mediaSterile distilled water96-well flat bottom microplate (e.g. Corning Cat. No. 3595) for imaging96-well round black round bottom ULA plate (e.g. Corning Cat. No. 45913799) or amber microtube (e.g. Cole Parmer Cat. No. MCT-150-X, autoclaved) for conjugation step0.01% Poly-L-Ornithine (PLO) solution (e.g. Sigma Cat. No. P4957), optional for non-adherent cells Recommended control antibodiesIt is strongly recommended that a positive and negative control is run alongside test antibodies and cell lines. For example, CD71, which is a mouse anti-human antibody, is recommended as a positive control for the mouse Fab.Anti-CD71, clone MEM-189, IgG1 e.g. Sigma Cat. No. SAB4700520-100UGAnti-CD71, clone CYG4, IgG2a e.g. BioLegend Cat. No. 334102Isotype controls, depending on isotype being studied—Mouse IgG1, e.g. BioLegend Cat. No. 400124, Mouse IgG2a e.g. BioLegend Cat. No. 401501Preparation of Incucyte® Antibody Internalization Assay 1. Seed target cells of interest1.1 Harvest cells of interest and determine cell concentra-tion (e.g. trypan blue + hemocytometer).1.2 Prepare cell seeding stock in target cell growth mediawith a cell density to achieve 40–50% confluence be-fore the addition of labeled antibodies. The suggested starting range is 5,000–30,000 cells/well, although the seeding density will need to be optimized for each cell type.Note: For non-adherent cell types, a well coating may be required to maintain even cell distribution in the well. For a 96-well flat bottom plate, we recommend coating with 50 μL of either 0.01% Poly-L-Or-nithine (PLO) solution or 5 μg/mL fibronectin diluted in 0.1% BSA.Coat plates for 1 hour at ambient temperature, remove solution from wells and then allow the plates to dry for 30-60 minutes prior to cell addition.1.3 Using a multi-channel pipette, seed cells (50 µL perwell) into a 96-well flat bottom microplate. Lightly tapplate side to ensure even liquid distribution in well. Toensure uniform distribution of cells in each well, allowthe covered plate sit on a level surface undisturbed at room temperature in the tissue culture hood for 30minutes. After cells are settled, place the plate insidethe Incucyte® Live-Cell Analysis System to monitor cell confluence.Note: Depending on cell type, plates can be used in assay once cells have adhered to plastic and achieved normal cell morphology e.g.2-3 hours for HT1080 or 1-2 hours for non-adherent cell types. Some cell types may require overnight incubation.2. Label Test Antibody2.1 Rehydrate the Incucyte® Fabfluor-pH Antibody Label-ing Reagent with 100 µL sterile water to result in a final concentration of 0.5 mg/mL. Triturate to mix (centrifuge if solution is not clear).Note: The reagent is light sensitive and should be protected fromlight. Rehydrated reagent can be aliquoted into amber or foilwrapped tubes and stored at -80° C for up to 1 year (avoid freezing and thawing).2.2 Mix test antibody with rehydrated Incucyte® Fabfluor–pH Antibody Labeling Reagent and target cell growth media in a black round bottom microplate or ambertube to protect from light (50 µL/well).a. Add test antibody and Incucyte® Fabfluor–pH Anti-body Labeling Reagent at 2X the final concentration.We suggest optimizing the assay by starting with afinal concentration of 4 µg/mL of test antibody or theFabfluor-pH Antibody Labeling Reagent (i.e. 2Xworking concentration = 8 µg/mL).Note: A 1:3 molar ratio of test antibody to Incucyte® Fabfluor-pHAntibody Labeling Reagent is recommended. The labeling re-agent is a third of the size of a standard antibody (50 and 150KDa, respectively). Therefore, labeling equal quantities will pro-duce a 1:3 molar ratio of test antibody to labeling Fab.b. Make sufficient volume of 2X labeling solution for50 µL/well for each sample. Triturate to mix.c. Incubate at 37° C for 15 minutes protected from light.Note: If performing a range of concentrations of test antibody,e.g. concentration response-curve, it is recommended to createthe dilution series post the conjugation step to ensure consistentmolar ratio. We strongly recommend the use of both a negativeand positive control antibody in the same plate.3. Add labeled antibody to cells3.1 Remove cell plate from incubator.3.2 Using a multi-channel pipette, add 50 µL of 2X labeledantibody and control solutions to designated wells.Remove any bubbles and immediately place plate in the Incucyte® Live-Cell Analysis System and start scanning.Note: To reduce the risk of condensation formation on the lid priorto first image acquisition, maintain all reagents at 37° C prior toplate addition.4. Acquire images and analyze4.1 In the Incucyte® Software, schedule to image every15-30 minutes, depending on the speed of the specific antibody internalization.a Scan on schedule, standard. If the Incucyte® Cell-by-Cell Analysis Software Module (Cat. No. 9600-0031)is available, adherent cell-by-cell or non-adherentcell-by-cell scan types can be selected.b Channel selection: select “phase” and “red” or“phase” and "orange” (depending on reagent used).c Objective: 10X or 20X depending on cell types used,generally 10X is recommended for adherent cells,and 20X for non-adherent or smaller cells.NOTE: The optional Incucyte® Cell-by-Cell Analysis SoftwareModule enables the classification of cells into sub-populationsbased on properties including fluorescence intensity, size andshape. For further details on this analysis module and its appli-cation, please see: /cell-by-cell.4.2 To generate the metrics, user must create an AnalysisDefinition suited to the cell type, assay conditions andmagnification selected.4.3 Select images from a well containing a positiveinternalization signal and an isotype control well(negative signal) at a time point where internalizationis visible.4.4 In the Analysis Definition:Basic Analyzer:a. Set up the mask for the phase confluence measurewith fluorescence channel turned off.b. Once the phase mask is determined, turn the fluores-cence channel on: Exclude background fluorescencefrom the mask using the background subtractionfeature. The feature “Top-Hat” will subtract localbackground from brightly fluorescent objects withina given radius; this is a useful tool for analyzing ob-jects which change in fluorescence intensity overtime.i The radius chosen should reflect the size of thefluorescent object but contain enough backgroundto reliably estimate background fluorescence inthe image; 20-30 μm is often a useful startingpoint.ii The threshold chosen will ensure that objectsbelow a fluorescence threshold will not bemasked.iii Choose a threshold in which red or orange objectsare masked in the positive response image but lownumbers in the isotype control, negative responsewell. For a very sensitive measurement, for example,if interested in early responses, we suggest athreshold of 0.2.NOTE: The Adaptive feature can be used for analysis but maynot be as sensitive and may miss early responses. If interestedin rate of response, Top-Hat may be preferable.Cell-by-Cell (if available):a. Create a Cell-by-Cell mask following the softwaremanual.b. There is no need to separate phase and fluorescencemasks. The default setting of Top-Hat No Mask forthe fluorescence channel will enable backgroundsubtraction without generation of a mask. Ensurethat the Top-Hat radius is set to a value higher thanthe radius of the larger clusters to avoid excess back-ground subtraction.c. The threshold of fluorescence can be determined inCell-by-Cell Classification.Specifications subject to change without notice.© 2020. All rights reserved. Incucyte, Essen BioScience, and all names of Essen BioScience prod -ucts are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. Publication No.: 8000-0728-A00Version 1 | 2020 | 04Sales and Service ContactsFor further contacts, visit Essen BioScience, A Sartorius Company /incucyte Sartorius Lab Instruments GmbH & Co. KGOtto-Brenner-Strasse 20 37079 Goettingen, Germany Phone +49 551 308 0North AmericaEssen BioScience Inc. 300 West Morgan Road Ann Arbor, Michigan, 48108USATelephone +1 734 769 1600E-Mail:***************************EuropeEssen BioScience Ltd.Units 2 & 3 The Quadrant Newark CloseRoyston Hertfordshire SG8 5HLUnited KingdomTelephone +44 (0) 1763 227400E-Mail:***************************APACEssen BioScience K.K.4th floor Daiwa Shinagawa North Bldg.1-8-11 Kita-Shinagawa Shinagawa-ku, Tokyo 140-0001 JapanTelephone: +81 3 6478 5202E-Mail:*************************5. Analysis GuidelinesAs the labeled antibody is internalized into the acidic environment of the lysosome, the area of fluorescence intensity inside the cells increases.This can be reported in two ways:Ways to Report Basic AnalyzerCell-by-Cell Analysis* To correct for cell proliferation, it is advisable to normalize the fluorescence area to the total cell area using User Defined Metrics.For Research Use Only. Not For Therapeutic or Diagnostic Use.LicensesFor non-commercial research use only. Not for therapeutic or in vivo applications. Other license needs contact Essen BioS cience.Fabfluor-pH Red Antibody Labeling Reagent: This product or portions thereof is manufactured under license from Carnegie Mellon University and U.S. patent numbers 7615646 and 8044203 and related patents. This product is licensed for sale only for research. It is not licensed for any other use. There is no implied license hereunder for any commercial use.Fabfluor-pH Orange Antibody Labeling Reagent: This product or portions thereof is manufactured under a license from Tokyo University and is covered by issued patents EP2098529B1, JP5636080B2, US8258171, and US9784732 and related patent applications. This product and related products are trademarks of Goryo Chemical. Any application of above mentioned technology for commercial purpose requires a separate li -cense from: Goryo Chemical, EAREE Bldg., SF Kita 8 Nishi 18-35-100, Chuo-Ku, Sapporo, 060-0008 Japan.SupportA complete suite of cell health applications is available to fit your experimental needs. Find more information at /incucyte Foradditionalproductortechnicalinformation,************************************************************/incucyte。
抗Xa试剂说明书
抗Xa试剂说明书使用说明医疗产品"用于测定直接抗凝剂抗Xa活性的试剂盒，适用于技术解决方案系列凝血仪"的使用说明。
任务TS-Anti-Xa"医疗设备用于在"技术解决方案"凝血仪上通过显色法对血浆中直接抗凝剂的抗Xa活性进行定量测定，以监测抗凝剂治疗情况，如非分数肝素和低分子量肝素、直接口服抗凝剂(POAC)（利伐沙班、阿哌沙班、磺达肝癸等）。
该试剂盒仅供实验室诊断领域的专业人员使用：实验室负责人、CLD医生、实验室医生、医学实验室技术员（辅助医务人员-实验室技术员）、实验室技术员、医学技术员。
试剂盒的应用领域：临床实验室诊断、临床医学。
试剂盒特点方法原理。
直接作用抗凝剂对凝血蛋白酶，特别是对Xa因子有抑制作用，因此具有抗血栓作用。
被测血浆样本中的抗凝剂会使Xa因子失活。
残留的Xa因子会特异性地水解显色底物，释放出硝基苯胺。
自动凝血仪记录405纳米波长处光密度随时间的变化，该变化与血浆中抗凝剂的浓度成反比。
在测定未分馏肝素和低分子量肝素的抗Xa活性时，应考虑到这种活性是由肝素与样本中的抗凝血酶复合物引起的。
这种复合物的活性取决于患者血浆样本中内源性抗凝血酶的水平。
因此，如果患者血浆样本中抗凝血酶水平较低，建议使用1号试剂盒中含有抗凝血酶的试剂盒。
试剂盒组成KitNo：1.因子-Xa试剂（冻干），每5ml-6fl.致色底物（冻干），每5毫升-3瓶。
分析试剂盒特点测定低分子量肝素和未分馏肝素的灵敏度-不超过0.05IU/ml(IU/ml)。
测定POAC的灵敏度--不超过10纳克/毫升（ng/ml）。
低分子量肝素和非分叶肝素测定的线性范围为0.05至1.0IU/ml（IU/ml），"线性"偏差不超过10%。
POAC测定线性范围为20至1000纳克/毫升（ng/ml），"线性"偏差不超过10%。
发现"试验--偏差不超过10%。
Sigma-Aldrich实验室常用生化试剂大促销
缓冲液
产品货号 英文品名 中文品名 优惠价 (R M B ) 目录价 (RMB)
A1542-2.5KG A1542-250G A1542-500G B7901-1KG B7901-500G C3041-100CAP C3041-50CAP C3674-100G C3674-1KG C3674-500G E9508-100ML E9508-10UL E9508-1L E9508-2.5L E9508-500ML E6758-100G E6758-500G H3375-100G H3375-1KG H3375-250G H3375-25G H3375-500G H3375-5KG I0125-100G I0125-10G I0125-1KG I0125-25G I0125-500G I0125-5KG M2933-100G M2933-1KG M2933-25G M2933-500G M1254-100G M1254-1KG M1254-250G M1254-25G M1254-50KG M1254-5KG P5493-1L P5493-4L P4809-100TAB P4809-50TAB
Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose Agarose
低熔点琼脂糖 低熔点琼脂糖 低熔点琼脂糖 低熔点琼脂糖 低熔点琼脂糖 低熔点琼脂糖 琼脂糖 琼脂糖 琼脂糖 琼脂糖 琼脂糖 琼脂糖
Ammonium acetate ~98% Ammonium acetate ~98% Ammonium acetate ~98% Boric acid Boric acid Carbonate-Bicarbonate Buffer Carbonate-Bicarbonate Buffer Citric acid trisodium salt Citric acid trisodium salt Citric acid trisodium salt Ethanolamine >=98% Ethanolamine >=98% Ethanolamine >=98% Ethanolamine >=98% Ethanolamine >=98% Ethylenediaminetetraacetic acid >=98.5% Ethylenediaminetetraacetic acid >=98.5% HEPES >=99.5% (titration) HEPES >=99.5% (titration) HEPES >=99.5% (titration) HEPES >=99.5% (titration) HEPES >=99.5% (titration) HEPES >=99.5% (titration) Imidazole >=98.5% (titration) Imidazole >=98.5% (titration) Imidazole >=98.5% (titration) Imidazole >=98.5% (titration) Imidazole >=98.5% (titration) Imidazole >=98.5% (titration) MES hydrate >=99.5% MES hydrate >=99.5% MES hydrate >=99.5% MES hydrate >=99.5% MOPS >=99.5% (titration) MOPS >=99.5% (titration) MOPS >=99.5% (titration) MOPS >=99.5% (titration) MOPS >=99.5% (titration) MOPS >=99.5% (titration) Phosphate buffered saline Phosphate buffered saline Phosphate-Citrate Buffer Phosphate-Citrate Buffer
【doc】一氧化氮合成酶（NOS）基因表达的半定量检测及其运用
一氧化氮合成酶（NOS）基因表达的半定量检测及其运用4生理,1994,4B(4),347354ActaPhysiologicaSinica一氧化氮合成酶(NOS)基因表达的半定量检测及其运用1张晨晖李肯虹,继峰周洪张新波场健r_元j五吾础研究所分子生物学研究室北京I.口舶3)-三Jl摘要'/4'本工作参照Mi~lin(1993)定量RTPeR方法,建立了一种灵敏,简捷,特异的定量NOSmRNA测定方法}证明了NOSmRNA不仅存在于脑组织,亦广泛分布于心,肾,肺和肝组织中,其中以脑组织含量最高,肾,心次之.除内皮细胞以外,平滑肌细胞中亦有NOS基因的表达}此外,本工作还观察到,自发性高血压大鼠的脑,肾,肝和平精肌细胞中NOSmRNA水平下降,提示NOS基因表达受抑,可能与高血压的病国密切相关.f上一,关键词:.=墨些窒盛堕差里室垄自发性高血压太最;半定量pcRzfl兰,-近年来的研究证明,一氧化氮(nitricoxide,NO)是一种血管内皮细胞释放的内皮衍化舒张因子(endothelium—delvedrelaxingfactors,EDRFs),它具有舒张血管,降低血压,抑制血管平滑肌细胞增殖和血小板粘附等重要的生理作用,在高血压,心肌缺血等许多心血管疾病的发病中具有重要意义.NO是由L广精氨酸(L—ARG)和分子氧在一氧化氮合成酶(nitricoxidesynthase,NOS)催化下合成的,NOS则是NO生成的关键酶.最近,NOS cDNA已经在大鼠内皮细胞,小脑中克隆出来0一:.但是关于它在体内不同组织的分布以及在心血管疾病高血压发病中的作用,迄今还未见报道.本工作应用聚合酶链式反应(po]y—merasechainreaction,PCR)技术建立了一种新的,简捷,特异的定量NOSmRNA的测定方法,研究了NOS在大鼠体内的分布,发现自发性高血压大鼠(spontaneoushyl~rtenslverat,SHR)脑,肾和肝中NOSmRNA的表达明显降低,其内皮依赖性的血管舒张反应亦明显下降提示,N0S基因表达下降,可能与高血压的病因密切相关.1材料和方法动物本工作选用8—1o周龄的雄性SHR和WKY(Wistar-Kyotorat,WKY)大鼠进行实验,SHR大鼠由Okamoto等选育成功,其特点为1oo%高血压自发率(BP=22.7士1.3kPa),同时具有高血压性心血管病变,适用于人类高血压病的研究,WKY为其正常对照模型,实验所用动物由中国医学科学院心血管研究中心提供车文19日3年6月8日收到1993年9月12日修回生理46卷试剂本工作所用的反转录酶(MoMLV)和TaqDNA聚台酶分别购自美国的Stret- gene公司和Perkin—Klmer公司,[P]dCTP为北京福瑞公司产品(放射比活度为3O00Ci/mmo1)RandomPrimer和dNTP购自美国Promega公司,其它试剂均购自美国Sigma 公司.细胞培养取用6—8周龄雄性SHR,WKY大鼠和新生小牛的胸主动脉段,分别按Hofman和Booyse的方法培养血管平滑肌细胞和内皮细胞.本实验选用5—8代细胞进行实验.总RNA的制备取10的平滑肌细胞或内皮细胞,用RNAzolBRNA提取试剂盒(美国TEL—TEST公司)提取细胞总RNA.各取1g脑,心,肺,肾,肝组织采用异硫氰酸胍一酚一氯仿方法:提取组织总RNA.总RNA提取完成后,分别用紫外分光光度计测定总RNA的浓度,重复测定三次.oD:OD.比值在1.8—2.0之间.计算样品总RNA的浓度.反转录(reversetranscription,RT)取2lag总RNA,在65℃变性3min,依次加入0.5mmol/LdNTP,0.01mol/LDTT,10pmol/LRandomPr/mer,15unit反转录酶和oH 8.350mmot/LTris—HCI,75mmol/LKCI,3mmol/LM鲁cl2缓冲液,总反应体积为201.在37℃保温90min.反应结束后,加入50TE缓冲液(pH8.010mmol/LTris—HCI,1mmol/ LEDTA),于70℃加热10min以终止反应定量PCRNOS寡核苷酸的引物合成是根据Bredt等E3]发表的大鼠小脑NoscD- NA序列应用DNA合成仪合成的.引物A,B序列分别为:5GG:GAATCCATACCAGCCTGATCCATGGAACC35TACTCGAAACGCCTGAA TGG3用这一对引物可以扩增出自2463bp-3124bp(661bp)的NOScDNA序列,长度相当于821aa一1041aaNOS氨基酸序列.取1/lo体积(71)反转录反应液,分别加入200mmol/LdNTP,50pmol/LNOS寡核苷酸引物,0.5ttCi32P]dCTP和pH8.310mmol/LTr/s—HCI.50mmol/LKC1,1.5mmol/ LMsCl缓冲液,2.5unitTaqDNA聚合酶;总反应体积为100l.最后加入50山轻矿物油.PCR反应所需的变性,退火和延伸温度分别为:94℃,55℃和72℃,反应时间分别为:1,2和3min.共进行25—35次循环.首次循环.94℃需要3min.反应完成后取10山PCR反应物进行1.5琼脂糖凝胶电泳(含0.5~tg/ml的溴化乙锭)分析,以0xl74/HaeⅢ为分子量标准,电泳缓冲液为1xTBE(0.09mol/LTris一硼酸,0.002mol/LEDTApH8.0). 电泳完毕后在紫外灯下照片,并切取含有荧光条带的凝胶,应用液体闪烁计数仪进行放射性测量.2实验结果2.1NosmRNA刹量方法的建立本工作依据Martin(1993)的方法建立了定量NOSmRNA测量方法.其结果如图1. 2所示.由图1可以看出,脑组织总RNA反转录后经25—35次PCR扩增后,其NOScDNA片段约为660bp与所设计的NOScDNA相同.其扩增量与扩增循环次数密切相关.由图2可以看出,脑组织总RNA样本量不同,RT—PCR后NOScDNA产率亦不同.具有明显的4期氧化氮合成酶(Nos)基因表达的半定量捡涮及其运用349剂量一效应关系;应用0.25ug的总RNA,即可检测到NOSmRNA圉1PCR扩增产率与循环次数之间的剂量一效应关系.1Dose—effectrelationshipbetwec"theyieldofPCRproductandthenumberofPCRampJificationcycle(n一3,:K2:SE).M:xl74/Ha~Il000Am0u"【ofIOtillRNA2530圉2PCR产量和总P,NA量之间的剂量一效应关系Fig.2D.se—effectrelationshipbetwoentheyieldofPCRproductandtheamountoftotalRNAn一3,士SE.0,l?2,3,4tamountoftOtalRNA(0,0t25,0.5,1.0,1.5.2,respec-tire竹).MI.xl74I/HaeI.…取浓度脑组织总RNA(2g),分别经过5RT—PCR后.NOScDNA产率基本相同?具有良好的重复性.结果见图3所示.圈32恒量总RNART-PCR重复实验Fjg?3RT-PCRrepeatexperimentwithaconstant~Jnount.ftotalRNA(2ug)l,2,3-4,5:RT—PCRrepeattimesM:似174/Ha~I.2?2N0SmRNA在大鼠体内分布取正常Wistar大鼠小脑,心,肺,肾,肝组织,提取总RNA,应用RT-PC~,测定不同组织中NOSmRNA的分布,其总RNA用量均为2.0ug,其结果如图4所示.由图可l}{∞一.r.....mr....L.................._-畜●吾j墨lu.u0u芍fd2自言q孙lE3u1lx,&5i0.I.0}dlu】lf【山生理46卷看出,在所测组织中均有NOSmRNA分布mRNA的含量约为肾脏和心脏的5倍.其中以小脑为最高,肝脏最低;其小脑内NOS取107的平滑肌细胞和内皮细胞,提取总RNA,经RT—PCT测定NOSmRNA,其细胞总RNA为2g.结果如图4所示.由图可以看出,NOSmRNA不仅存在于内皮细胞,亦分布于平滑肌细胞中,内皮细胞NOSmRNA的含量较高于平滑肌细胞图4NOS基因在正常大鼠不同组织和培养细胞中的表选Fig.4ExpressionofNOSgeneinvarioustissuesandculturedcellsofnormalratby RT—PCRM:HX174/HaeI}H:Heart;B}Brain;P;Lung;E,EC:Endothelialcall;K:KJdney~L!Uver;S,SMC:Smoothmusclec~11.2.3自发性高血压大鼠NosmRNA的变化分别取SHR和WKY大鼠的小脑,肾,肝组织,提取总RNA,依前法测定NOSmRNA .其所测样品总RNA用量均为2.结果如图5所示,由圈可以看出,在所检测的组织中,SHR大鼠NOSmRNA均低于WKY正常大鼠,其中在小脑降低了39.在肾脏降低了55,在肝脏降低了88.分别取10T个培养的WKY和SHR主动脉平精肌细胞,提取总RNA,各取2,同样用RT-PCR检测NOSmRNA,结果如图5所示.由图亦可以看出,SHR大鼠平精肌细胞中NOSmRNA含量明显降低,约较WKY大鼠降低了62.由以上结果可以看出,在SHR大鼠,无论是平滑肌细胞还是小脑,肾脏,其NOS基因的转录均明显降低.2.4自发性高血压大鼠内皮依赖性血管舒张反应为了进一步鉴定SHR大鼠血管EDRF/NO的反应性.取6只SHR大鼠主动脉环进行离giJc05u'j.三nlJ|.;4期一氧化氨合成酶(NOS)基因表达的半定量检测及其运用35J团SHR口WKY图5NOS基因在sHR和WKY不同组织和细胞中的表达Fig.5ExpressionofNOSgeneinvarioustissuesandculturedcellsinSHRandWKYn一3,土sE;P<0.05,.'P<0.01.M:174/HaeISMC:SmoothmusclecellSc:SHRSMC}SL:SHRLung;SK{SHRKidney;SB:SHRBrain;We!WKYSMClWL:WKYLung;WK:WKYKidney'WB:WKYBrain-圈6乙酰胆碱诱导的SHR和WKY主动脉内皮依赖性舒张反应≤Fig-6Endothelium—dependentrelaxationevkedbyAChinaortafromSHRandWKY.n=6.~4-SE,P<0.05,"P<0.01.j0——0WKygroupI●——●跚Rgroup1——L-ARG+SHR.囊～体灌流实验,观察乙酰胆碱(ACh)内皮依赖性血管舒张反应,结果如图6所示.由图可以看出.SHR大鼠主动脉ACh所引起的血管内皮依赖性舒张反应明显低于WKy 大鼠,其㈣uIdI.iirJ0j△2EⅢl;=E生理46卷最大的舒张反应亦只有WKY大鼠的I/2.为了进一步验证SHR大鼠ACh内皮依赖性血管舒张反应降低的机制,本实验还将10mol/LL-ARG预先加入SHR大鼠主动脉环灌流槽中,以补充EDRF/NO的前体,10min后,再进行ACh舒张反应实验,结果如图6所示.由图可以看出,补充Ⅱ)F,NO的前体,可使SHR主动脉ACh内皮依赖性舒张反应恢复.这些实验结果说明,自发性高血压大鼠NOS基因转录和表达下降,内皮依赖性血管舒张反应降低.3讨论3.1mRNA的测定mRNA的测定是分子生物学研究中一个常用的方法,主要有斑点杂交,NorthernBlotting等几种方法].但是由于mRNA含量少,又极易降解,因此对于其含有极小量特异mRNA的组织和样品,测定较为困难为此,近年来发展了RNA—protoc-fiveassay和含内标化的RT—PCR定量测定mRNA的方法.RNA—protectiveassay 虽然灵敏,准确,但操作复杂,价格昂贵,不易普及].定量RT—PCR虽然简便,快速并能定量,但因含有一个"内标准"而易产生"管效应",从而抑制特异性RT—PCR反应,干扰mRNA 的测定].本工作参照Martin(1993)~所建立的RT-PCR定量测定p-肾上腺素能受体mRNA 的方法,建立了NOSmRNA的测定方法.这种方法不另设内标准,排除了"管效应",而应用["P3dC'FP直接掺入PCR扩增反应,这不仅可使小量mRNA通过RT—PCR得以扩增和放大,而且可以通过掺入的放射性强度,直接进行rnRNA的定量测定.这种方法具有明显的荆量一效应关系和良好的重复性.我们曾经应用NorthernBlotting方法,测定不同组织中NOSmRNA含量,结果发现需要4O一6O嵋总RNA,才可检测到太鼠小脑和肾中NOSmR-NA,在肝脏,肺,平滑肌细胞中即使应用再太剂量总RNA亦难以测定;而应用本文这种方法,只需0.25—2g总RNA,即可测出组织中所含的NOSmRNA.应该指出.应用这种方法时,对提取的总RNA定量必须十分准确.ODOD的比值必须在18以上,总RNA定量应多次测量.3.2NOSmRNA在体内分布关于NOSmRNA在组织中的分布已有报道Brcdt等(1991)应用大鼠脑NOSeDNA探针和NorthernBlotting分析,发现NOSmRNA只存在于脑内rIWilliam等(1992)应用牛内皮细胞NOSeDNA为探针和NorthernBlotting分析,发现NOS只分布于内皮细胞中0}从而提出NOSmRNA不是普遍存在的.在许多组织中很难测到NOSmRNA的存在,但是不同组织的NOSe.DNA具有高度同源性[JI应用NOS 生物测定的方法证明,N0s亦广泛分布于不同组织中口".因此我们设想,在一些组织中难以测定出NOSmRNA,主要是因为组织中NOS特异性的mRNA在总RNA中所占比例过少,测定方法不够灵敏所致.本工作应用快速,灵敏的定量RT-PCR方法,不仅测出了脑内和内皮细胞中NOSmRNA,亦测出平滑肌细胞和心脏,肾脏,肺,肝脏组织中NOSmRNA.提示:NOSmRNA在体内不同组织中是广泛存在的.这与生物检测的结果相一致.过去认为NOSmR—NA主要存在于内皮细胞和巨噬细胞,本工作证明了在平滑肌细胞中亦有NOS的高表达}最近Nunokawa等(】993)从平滑肌细胞中克隆到NOSeDNA:Ⅲ,说明平滑肌细胞和内皮细胞4期一氧化氮合成酶(NOS)基因表达的半定量检测及其运用353一样,亦是NOS基因转录和表达的场所之一.近年来的研究表明,NOS可分为结构型和诱导型两类,而大鼠脑NOS即属于结构型[t3;我们应用依据大鼠脑NOScDNA序列设计合成的PCR引物,检测出在脑,心,肾,肺,肝,血管内皮细胞和平滑肌细胞中都有NOSmRNA的表达提示,结构型NOS可能是不同组织中所共有的.关于NOS结构型与诱导型之间的相互关系及其组织特异性,还需进一步研究3.3NOS在高血压发病中的作用既往的研究证明,内皮细胞损伤,Ea~RF/NO生成障碍是高血压发病的一个重要因素口.我们的实验亦证明,SHR大鼠血管内皮依赖性舒张反应明显下降.但是,这些研究都是基于药理方面的实验或是间接测定NOS的结果.关于高血压时,NOS基因转录和表达的变化,迄今尚未见报道.我们首次测定了SHR 大鼠不同组织和平滑肌细胞中NOSmRNA的变化,发现SHR大鼠NOS基因转录和表达明显低于WKY大鼠NOS是EDRF/No生成的关键因素,NOS基因表达下降,可致EDRF/NO 生成下降,这可能是高血压发生和发展的又一个重要因素.关于高血压时NOS基因转录下降的机制目前尚不了解.现有一些实验证明,NOS 基因表达受许多生长园子,细胞园子的调节Its].因此SHR大鼠NOSmRNA下降,亦可能是继发于生长因子,细胞因子的变化,亦可能是基因结构的改变所致.我们的初步实验证明,应用NOScDNA探针对SHR大鼠NOS基因进行限制性长度多态性分析,发现SHR 大鼠NOS基因可能有多态性变化.参考文献[1]FrinC~-R.GandOanso~,nL(1993).Pdsinginterest_mnitricoxidesyntha~.T/B8,18(2), 35—36.[2]William,C.S.,Jeffrey,K.H.andCynil~,M.B.(1992).Molecularcloningandexpressionof aeDNAencodingendot~lialcellnitricoxidesymha~.,J.嘲.0.,2I1,15274—15276.[3]Brech-nS.-Hwang,P.andSnyde,S.H.(1991).Clonedandexpre~ednitricoxidete$ntlm~s ttu~-rurallyre穹embl鹪~ytoeaxrocnoP-450reductase.神,351,7l4—718.[4]Herman.W.andGooSer,D.(1977).hmmn0fIucenoeintheidentificationofdtfferer*ttattnamoothmugcleoe1lsinculture.脚.D妇H.朋啪H∞-18,52—54.[5]Booyse,F.M.,Sedlak,B.J.andRatelson,E.(1975).Ctlltttre0farterialend劬e1iaIcellslcharacterl-zat~onandgrowthofbovineaofticendothelJalceils.M帕.姗商靖∞..¨.925—939. [6]aI∞mii1|H,Rand鼬ochl,N.(1987).Sin~e-stepmethod0P2qAIscdailonbyacidguantdiniumcain- extraction.肺c_Blsd~a..162.156—159.[7]Mar吐n,U..Michael,B.andJohn,S.E.(1993).Altered蜘酋onB-sdr能舡g【te憷呻rkimuleand~-adeene.zglc胁D佃reinthetatartshumanheart.帕'曲_,87,454—463.[8]Frederick-M.A.,Roge~,h-Robert,E.K.,Dav~l,nM.,S~idman,J.G.,dehn+A.S.and Kcvln,S.(1992).丽州丹咄帅缸脚咖.PlIb1hedbyGreenePublishingAssociatesandJohn Wiley&Sons.,2rid.-呻.4-18～4-23.NewY ork.[93A1ice,M.W.,M~baeJ,V.D.andDavid,F.址(1989).QI衄乜吐onmRNAbymeIxdymeras~chainteac~on.Neff..删...86.9717—9721.[10]Natlum,C.(1992).Njn缸oxideasasecretorypr~luetofmammaliancells.FA~BBJ.,8,3051—3064.[¨]Hchalet"son,A.H.(1991).FMldothelinminCOllt/e1..^J..B5,ll6—125.[12]Nunol~wa?Y.,Islti~a-N.andTana[~,&(1993).Cloningofinducibleninjcoxidesy nthas~r砒v-~lscu—lar~atooth/~rluseleo~ils..咖.脑."矾嘲.,191-89—94,生理46卷[13]David,A.G.,Andreas,K.N.andMauriclo,D.S.(1993).Cytokines,endotoxin,andgluco corticoidsregulatetheexpressionofinduciblenixieoxidesyntha~inhepatocytes.Proc.NatI_删?U3A.?90,施新猷(1989).医学实验动物学PP.39—40.陕西科学出版社,西安. ActaPhysio~ogicnSinlca1994,4g(4),347—354 SEMIQUANTITATIVEDETECTIONANDAPPLICATIONABOUT THEEXPRESS10NOFNIT砒COXIDESYNTHASEGENEZHAN6CHEN-HUI,LIQlANHONG,ZHAN6JIFNG,ZHOUHONG, ZHANGXn~G-BOANDTANGJIAN(,Mdeen~丑乩鲫,Card~sedar8硒由,&咖Ma~a/,&i谛10083)A±I'RAClUsingtheReverseTranscription(RT)一PolymeraseChainReaction(PER)method ofMartin(1993)forsemiquantitationdeterminatingofNOSgene,itwasfoundthat NOSmRNAisnotonlyexistedinbrain.butalsodistributedextensivelyinheart,kid- hey,lungandliver.Amongofthem,NOSmRNAlevelswerehighestinthebrain'fol- lowedindescendingorderbykidney,heart.Inadditiontoendothelialcell,NOSgene wasalsohighlyexpressedinsmoothmusclecells,suggestingthattheymaybeaDimpor- tantsiteofNOSinorganism.Furthermore.NOSmRNAlevelswerefoundtodecrease significatelyinbrain,kidney,liverandsmoothmusclecellinspontaneoushypertensive rat.Thesedatasuggestthatpathogenyofbypertentionmayberelatedtolowexpression 0fNOSgeneinthesetissues.KeywordstnitricoxideJv/nthm}8eneexpre*tton{polymerase~[111111reaction。
Elabscience 花生四烯酸(AA)酶联免疫吸附测定试剂盒使用说明书
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Copyright ©2021-2022 Elabscience Biotechnology Co.,Ltd. All Rights Reserved目录用途 (3)基本性能 (3)检测原理 (3)试剂盒组成及保存 (4)试验所需自备物品 (5)样品收集方法 (5)注意事项 (6)■ 试剂盒注意事项 (6)■ 样品注意事项 (6)样本稀释方案 (6)检测前准备工作 (7)操作步骤 (8)结果判断 (10)技术资源 (10)典型数据 (10)性能 (11)■ 精密度 (11)■ 回收率 (11)■ 线性 (11)声明 (12)Intended use (13)Character (13)Test principle (13)Kit components & Storage (14)Other supplies required (15)Sample collection (15)Note (16)■ Note for kit (16)■ Note for sample (16)Dilution Method (17)Reagent preparation (17)Assay procedure (18)Calculation of results (20)Technical resources (20)Typical data (20)Performance (21)■ Precision (21)■ Recovery (21)■ Linearity (21)Declaration (22)用途该试剂盒用于体外定量检测 血清、血浆或其他相关生物液体中AA浓度。
非放射性凝胶迁移试剂盒（EMSA）说明书
（1）湿转
1） 配制电转印缓冲液：配制电转印缓冲液0.5×TBE 1200ml。
10×TBE 双蒸馏水 总量
60ml 1140ml 1200ml
电转印可在室温下进行。
2） 浸膜：将结合膜，2张厚滤纸和电转印泡沫垫在0.5×TBE浸泡10分钟（注意标记加样顺序）。
3） 装配电转印夹：电泳后，慢慢从凝胶上移走一片玻璃，将凝胶与另一片玻璃一起移入装有 TBE的盒中，在让凝胶与玻璃分离，使凝胶漂浮在TBE中。取一片预湿的滤纸在TBE中移动 到凝胶板下面，小心将滤纸与凝胶对齐一起从TBE中移出，置于电转印泡沫垫上并按图示的 顺序装配电转印夹。注意凝胶板与结合膜以及与滤纸之间不能有气泡。
脱气10min 10% AP（过硫酸胺） 总量
100µl 20.0ml
注:以上组分为制作两片70×80×1.5mm聚丙烯酰胺凝胶的用量，仅供参考。
（2） 制备预冷的0.25×TBE，4ºC保存:
10×TBE 双蒸馏水 总量
30ml 1170ml 1200ml
（3） 预电泳：用预冷的0.25×TBE在冰上120V 预电泳1小时。并在上样前用电泳缓冲液冲洗 加样孔3－5次/孔。
4） 电转印：将装配好的电转印夹放入电转印槽中，在0.5×TBE中，390mA电转印40分钟。注意 电转印的电极方向不能弄错。
（2）半干转印
以美国Bio-rad（伯乐）Trans-Blot SD半干转印仪为例
1） 配制电转印缓冲液：配制电转印缓冲液0.5×TBE 200ml。
10×TBE 双蒸馏水 总量
（4） 膜洗涤： 制备1×洗涤液：
5×洗涤液 双蒸馏水 总量 (15ml×4)
12ml 48ml 60ml
10ml 190ml 200ml
抗凝血因子XI抗体说明书
(19)国家知识产权局(12)发明专利申请(10)申请公布号 (43)申请公布日 (21)申请号 202111467365.1(22)申请日 2018.08.09(62)分案原申请数据201880098606.X 2018.08.09(71)申请人 上海仁会生物制药股份有限公司地址 201321 上海市浦东新区周浦镇紫萍路916号(72)发明人 王文义 于泉 刘小五 徐立忠 杜治强 (74)专利代理机构 北京润平知识产权代理有限公司 11283专利代理师 刘依云 刘亭亭(51)Int.Cl.C07K 16/36(2006.01)A61K 39/395(2006.01)A61P 7/02(2006.01)C12N 15/85(2006.01)C12N 5/10(2006.01) (54)发明名称抗凝血因子XI抗体(57)摘要本技术公开了结合凝血因子XI(FXI)和/或其活化形式因子XIa(FXIa)，或FXI和/或FXIa的片段的抗体，以及含有该抗体的组合物。
还公开了制备抗体的方法，以及抗体用于制备治疗和/或预防凝血相关病症(例如血栓和与血栓相关的并发症或病症)药物的用途。
权利要求书1页 说明书36页序列表104页 附图18页CN 114478782 A 2022.05.13C N 114478782A1.一种分离的抗FXI或抗FXIa抗体，其特异性结合人FXI或FXIa，其中所述抗体包含选自SEQ ID NO：155‑160的CDR的组合，具有至少90％同一性的序列，或其免疫活性部分。
2.根据权利要求1所述的抗体，其中所述的抗体特异性结合人FXI或FXIa的A3结构域。
3.根据权利要求1所述的抗体，其中，所述抗体包含选自以下序列对的一对序列：SEQ ID NO：153‑154，SEQ ID NO：201‑202，和SEQ ID NO：204‑205，以及具有至少90％同一性的序列。
4.一种药物组合物，其包含权利要求1‑3中任一项所述的抗体。