生物素化试剂盒-Biotinylation Kit

生物素标记试剂盒使用说明书货号: EBLK0002产品介绍：Elabscience生物素标记试剂盒提供了生物素标记所需全部试剂，用于含有氨基（NH2-）抗体的标记。

生物素已经活化，可直接使用，每个试剂盒足以完成3次标记，每次可标记0.2-2mg。

试剂盒中包括6个用于抗体标记脱盐的Filtration tube，不用透析，操作简便，熟练操作90min可完成整个标记过程。

产品特点：试剂全面:本试剂盒提供了生物素标记所需全部试剂。

快速:整个过程仅需90min。

方便:通过Filtration tube即可脱盐，无需透析或者凝胶过滤。

使用灵活:既可用于微量标记又可大量标记，每次可标记0.2-2mg。

理想的标记效果:已经优化确定了最适的标记比例，降低标记不足或由于过度标记而失活的可能性。

产品组成：标记过程需要仪器：1. 10ul，50ul，200ul，1000ul可调高精度移液器2. 恒温箱（37℃）3. 离心机（离心力可达到12,000×g）储存条件：本试剂盒未开封前在2-8℃可稳定保存一年生物素标记反应原理：NH2-Reactive Biotin专一地与伯胺反应（N-末端及赖氨酸残基侧链）形成稳定的酰胺键生物素标记NH2-Reactive Biotin使用量的计算：每个反应中生物素试剂的使用量取决于待标记蛋白质的量和浓度。

通过优化，我们确定了标记2mg/ml的抗体（IgG ，150KD），使用生物素和抗体的分子比为20:1能达到较理想的标记效果。

1、标记2mg/ml的抗体，使用生物素和抗体的分子比为20:1时，应加入生物素量的计算方法：ml蛋白×2mg蛋白ml蛋白×1mmolIgG150,000mgIgG×20mmol生物素mmol蛋白= mmol生物素2、对于10mmol的生物素溶液，应加入反应中该生物素体积的计算方法：mmol生物素×1,000,000μLL ×L10mmol= ul生物素计算示例：对于0.5ml 2mg/ml的IgG（分子量为150,000）溶液，需加入10mM的生物素溶液13.3ul。

化学发光法生物素标记核酸检测试剂盒产品编号 产品名称包装 D3308化学发光法生物素标记核酸检测试剂盒1000cm 2产品简介：化学发光法生物素检测试剂盒(Chemiluminescent Biotin-labeled Nucleic Acid Detection Kit)是一种通过Streptavidin-HRP 及后续的BeyoECL Moon 试剂来实现化学发光检测Biotin 标记核酸的检测试剂盒。

适用于Southern blot 、Northern blot 、ribonuclease protection assay (RPA)或EMSA 等实验中，采用生物素标记的DNA 或RNA 探针时的检测。

本试剂盒不适用于生物素标记蛋白的检测。

本试剂盒同时还提供了封闭液、洗涤液等检测时所需的配套试剂。

本试剂盒采用了高质量的Streptavidin-HRP Conjugate ，HRP 和Streptavidin 共价交联的比例大于3，这样比采用Streptavidin 和Biotin-HRP conjugate 两种试剂进行检测要更方便，并且灵敏度更高。

采用了非特异性结合比avidin 更低的strepatavidin ，使检测结果背景更低灵敏度更高。

本试剂盒没有提供生物素探针标记相关的试剂，生物素标记的DNA 探针或EMSA 探针的制备可以相应地使用碧云天生产的生物素3'末端DNA 标记试剂盒(D3106)或EMSA 探针生物素标记试剂盒(GS008)。

本试剂盒可以用于检测至少10块10×10cm 有生物素标记EMSA 探针的膜，即共1000cm 2。

包装清单：产品编号 产品名称包装 D3308-1 BeyoECL Moon A 液 55ml D3308-2 BeyoECL Moon B 液 55ml D3308-3 Streptavidin-HRP Conjugate100µl D3308-4 封闭液 380ml D3308-5 洗涤液(5X)250ml D3308-6检测平衡液 250ml —说明书1份保存条件：D3308-3 Streptavidin-HRP Conjugate 在-20ºC 保存，其余可4ºC 保存。

碧云天生物技术/Beyotime Biotechnology 订货热线：400-168-3301或800-8283301 订货e-mail ：******************技术咨询：*****************网址：碧云天网站 微信公众号Human IL-1β ELISA Kit产品编号 产品名称包装 PI305Human IL-1β ELISA Kit96次产品简介：碧云天的Human IL-1β ELISA Kit (Human Interleukin-1β Enzyme-Linked ImmunoSorbent Assay Kit)，即人白细胞介素1β酶联免疫吸附检测试剂盒，是一种用于特异性地高灵敏地定量检测人血清、血浆、细胞或组织裂解液、或细胞培养上清液中的IL-1β的ELISA 试剂盒。

本产品检测灵敏度高，特异性强，重复性好。

多次重复检测结果表明，最小检出量为2.2pg/ml ，与人IL-1r α、IL-1sRII 、IL-1sRI 、小鼠IL-1α、小鼠IL-1β等均没有交叉反应，板内、板间变异系数均小于10%。

IL-1即白细胞介素-1(简称白介1)，其家族由IL-1α(也称IL-1F1)、IL-1β(也称IL-1F2)、IL-1受体拮抗剂(IL-1 receptor antagonist, 简称IL-1RA, 或IL-1F3)及IL-18、IL-33和IL-1F5~F10组成。

IL-1主要由巨噬细胞产生，此外几乎所有的有核细胞，如B 细胞、NK 细胞、体外培养的T 细胞、角质细胞、树突状细胞、星形细胞、成纤维细胞、中性粒细胞、内皮细胞以及平滑肌细胞均可产生IL-1。

正常情况下只有皮肤、汗液及尿液中含有一定量的IL-1，绝大多数细胞在受到外来抗原或细菌内毒素刺激后才能合成和分泌IL-1。

IL-1β在免疫和炎症反应、骨重建(bone remodeling)、发烧、碳水化合物代谢等生理、病理过程中都有重要作用。

生物素化抗体使用说明
Biotin Antibody Conjugates
规格：0.1ml
保存：-20℃保存至少一年，避免反复冻融。

4℃保存3-6个月。

产品说明：
生物素化抗体是经亲和层析纯化成高纯度IgG成分，将进口的活化生物素（羟基琥珀酰亚胺酯，BNHS）共价交联到抗体IgG分子上制成。

生物素化抗体标记的样品可应用酶标或荧光染料标记的亲和素或生物素-亲和素复合物来检测。

本产品0.1ml含有0.2mg抗体，0.02mgBNHS，0.3mgBSA。

工作效价：
ELISA法：1：500～1000
组织化学：1：50～100
免疫印迹：1：200～500。

具体效价以产品标签为准，最佳使用条件需根据所检测样本的抗原及一抗效价优化而定。

产品说明：
SP034兔IgG免疫球蛋白抗原
SPA131羊抗小鼠IgG(纯化)
SF134羊抗兔IgG-FITC
SE237兔抗猪IgG-HRP
A1820HRP标记的抗体稀释液。

客户使用Apexbio产品发表的文献COA (Certificate Of Analysis)NMR (Nuclear Magnetic Resonance)MSDS (Material Safety Data Sheet)SDF溶解性H2O: ≤2 mg/mL with sonication储存条件Store at 4°C一般建议为了使其更好的溶解，请用37℃加热试管并在超声波水浴中震动片刻。

储液可以在零下20℃中保存数月。

运输条件试用装：蓝冰运输。

其他可选规格：常温运输或根据您的要求用蓝冰运输。

生物活性描述NHS-LC-Biotin是一种胺反应性的生物素化试剂。

靶点IC50产品描述NHS-LC-biotin（succinimidyl-6-(biotinamindo)hexanoate），也被称为NHS-X-biotin，是D-生物素的衍生物，一种胺反应性的生物素化试剂，由D-生物素的戊酸侧链通过间隔臂与NHS酯基团连接而形成。

NHS-LC-biotin末端的NHS酯基与蛋白或其它分子上的胺基共价结合形成稳定的酰胺键，并释放NHS基团。

NHS-LC-biotin的6-氨基己酸间隔臂极大地增加了共价修饰分子与双环生物素环之间的长度，导致对亲和素或链霉素探针更好的结合潜力。

NHS-LC-biotin在水溶液中是不溶的，在加入缓冲反应之前需先溶解于有机溶剂中。

参考文献：Bioconjugate Techniques , 2nd ed. By Greg T.Hermanson (Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL). Academic Press (an imprint of Elsevier): London, Amsterdam, Burlington, San Diego . 2008. ISBN 978-0-12-370501-3.。

INSTRUCTIONSNumberDescription28005 Kit Contents:24 microtubesBiotinylated Horseradish Peroxidase(40kDa), 5mgStorage: Upon receipt store at 4°C. Product is shipped at ambient temperature.Introduction4-Biotin (Product No. 21329). HABA (4´-hydroxyazobenzene-2-carboxylic acid) is a reagent that enables a quick estimation of the mole-to-mole ratio of biotin to protein. The Thermo Scientific™ Pierce™ Biotin Quantitation Kit contains a premix of HABA and avidin and a biotinylated horseradish peroxidase (HRP) positive control. The HABA/Avidin Premix is supplied in convenient Thermo Scientific™ No-Weigh™ Microtube packaging, which eliminates the difficulties associated with weighing small quantities of reagent.the protein. A protein can be conjugated with several biotin molecules, each of which can bind one molecule of avidin, thereby greatly increasing the sensitivity of many assays. The bond formation between biotin and avidin is rapid and once formed is unaffected by most extremes of pH, organic solvents and other denaturing agents.1-3To quantitate biotinylation, a solution containing the biotinylated protein is added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces the HABA and the absorbance at 500nm decreases proportionately. By this method, an unknown amount of biotin present in a solution can be quantitated in a single cuvette by measuring theabsorbance of the HABA-avidin solution before and after addition of the biotin-containing sample. The change in absorbance relates to the amount of biotin in the sample by the extinction coefficient of the HABA-avidin complex.Important Product Information• Following any biotin-labeling reaction, the biotinylated protein sample to be assayed must be dialyzed or desalted to remove nonreacted and hydrolyzed biotinylation reagent.•Samples must be in one of the recommended buffers (PBS or TBS, see Reagent Preparation Section) for the assay. Avoid buffers containing potassium (such as Modified Dulbecco’s PBS), which will cause precipitation in the assay. Other buffers may interfere and should not be used unless first validated by comparison to results using PBS or TBS. •Slight color variation between the HABA/Avidin Premix microtubes does not affect product performance.(Product No. 28376)Note: Avoid using buffers containing potassium salts (see Important Product Information).Biotinylated HRP (Positive Control)Visit our website to obtain the lot-specific certificate of analysis for the kit (Product No. 28005); note the exact package size (5 to 6mg) of the supplied Biotinylated HRP. Prepare the Biotinylated HRP at 1mg/mL by adding the appropriate volume of ultrapure water to the vial. Mix with a pipette tip and allow it to solubilize. Complete solubilization requires approximately 5 minutes at room temperature. Store solution in single-use volumes (i.e., 120μL ) at -20°C until ready to use.Pierce Biotin Quantitation KitProcedure for Quantitation of Moles of Biotin per Mole of Protein – Cuvette Format Note: The Biotinylated HRP may be used as a positive control to verify assay performance. See the product label for the biotinylation level.1.Equilibrate the HABA/Avidin Premix to room temperature.2.Add 100μL of ultrapure water to one microtube of the HABA/Avidin Premix. Mix with pipette tip.3.Pipette 800μL of PBS or other sample buffer into a 1mL cuvette. Use this cuvette with PBS to zero thespectrophotometer.4.Add the 100μL of the HABA/Avidin Premix solution from step 2 to the cuvette. Mix by inversion.5.Measure the absorbance of the solution in the cuvette at 500nm and record the value as A500 HABA/avidin.6.Add 100μL of biotinylated protein sample or biotinylated HRP (positive control) to the cuvette containing HABA/avidinand mix well.7.Measure the absorbance of the solution in the cuvette at 500nm and record the value as A500 HABA/avidin/biotin sampleonce the value remains constant for at least 15 seconds. If the A500 HABA/avidin/biotin sample is ≤ 0.3, dilute the sample and repeat the assay.Note: Dilutions must be accounted in the calculation step.8.Proceed to the Calculation of Moles of Biotin per Mole of Protein section.Procedure for Quantitation of Moles of Biotin per Mole of Protein – Microplate Format Note: The Biotinylated HRP may be used as a positive control to verify assay performance. See the product label for the biotinylation level.1.Equilibrate the HABA/Avidin Premix to room temperature.2.Add 100μL of ultrapure water to one microtube of the HABA/Avidin Premix. Mix with pipette tip.3.Pipette 160μL of PBS into a microplate well.4.Add 20μL of the HABA/Avidin Premix solution from step 2 to the PBS in the well. Place microplate on an orbital shakeror equivalent to mix.5.Measure the absorbance of the solution in the well at 500nm and record the value as A500 HABA/avidin.6.Add 20μL of biotinylated sample or Biotinylated HRP (positive control) to the well containing the HABA/avidinreaction mixture. Mix as described above.7.Measure the absorbance of the solution in the well at 500nm and record the value as A500 HABA/avidin/biotin sampleonce the value remains constant for at least 15 seconds.8.Proceed to the Calculation of Moles of Biotin per Mole of Protein section.Calculation of Moles of Biotin per Mole of ProteinNote: The HABA Calculator, which is available from the Technical Resources menu from our website, will calculate the moles of biotin/mole of protein upon entering the required values.These calculations are based on the Beer Lambert Law (Beer’s Law): Aλ = ελ bCWhere:A is the absorbance of the sample at a particular wavelength (λ). The wavelength for the HABA assay is 500nm.There are no units for absorbance.εis the absorptivity or extinction coefficient at the wavelength (λ). For HABA/avidin samples at 500nm, pH 7.0extinction coefficient is equal to 34,000 M-1cm-1.b is the cell path length expressed in centimeters (cm). A 10mm square cuvette has a path length of 1.0cm. Using therecommended microplate format volumes, the path length is typically 0.5cm.C is the concentration of the sample expressed in molarity (= mol/L = mmol/mL).The values needed for calculating the number of moles of biotin per mole of protein or sample are as follows: • Concentration of the protein or sample used, expressed as mg/mL• Molecular weight (MW) of the protein, expressed as grams per mole (e.g., HRP = 40,000; IgG =150,000) • Absorbance at 500nm for HABA/avidin reaction mixture (A 500 H\A )• Absorbance at 500nm for HABA/avidin/biotin reaction mixture (A 500 H\A\B )•Dilution factor, if the sample is diluted before adding to the HABA/avidin reaction mixture1. Calculation #1 is for the concentration of biotinylated protein in mmol/mL (before any dilution for the assay procedure):1#(mg/mmol)protein of MW (mg/mL)ion concentrat protein mL per protein mmol Calc ==2. Calculation #2 is for the change in absorbance at 500nm:• Cuvette:∆A 500 = (0.9 × A 500 H\A) – (A 500 H\A\B) = Calc #2 • Microplate:∆A 500 = (A 500 H\A) – (A 500 H\A\B) = Calc #23. Calculation #3 is for the concentration of biotin in mmol permL of reaction mixture:3#)(34,0002#)(34,000ΔA mixture reaction ml biotin mmol 500Calc b Calc b =×=×=4. Calculation #4 is for the mmol of biotin per mmol of protein:sampleoriginal in protein mmol sampleoriginal in biotin mmol =sample original in protein ml per mmol factor)(dilution )10mixture)(reaction in biotin ml per (mmol =1factordilution 10)3#(Calc#Calc ××=TroubleshootingAdditional InformationA.Optional Pronase DigestionNote: Pronase can be used to digest the protein to expose biotin groups that may be buried within the molecule and sterically hindered from binding to avidin. This Pronase method is optional and is not normally necessary because for most applications it is sufficient to quantify the number of biotin groups available on the surface of the protein molecule.1.Prepare 1% Pronase (w/v) in ultrapure water.2.Heat 100µL of biotinylated protein sample at 56°C for 10 minutes.3.Add 10µL of 1% pronase to the sample and digest overnight at room temperature.4.Quantitate biotinylation as previously described.B.Please visit our website for additional information on this product including the following:•Use the HABA Calculator to determine the moles of biotin/mole of protein in your sample. Enter the absorbance values, protein molecular weight and protein concentration and the biotin/protein molar ratio will be calculated for you. The HABA Calculator can be accessed from the Technical Resources menu.Related Thermo Scientific Products21329 EZ-Link NHS-PEG4-Biotin, No-Weigh Format, 8 × 2mg21329 EZ-Link NHS-PEG4-Biotin, 25mg21331 EZ-Link Sulfo-NHS-SS-Biotin, 100mg21334 EZ-Link Iodoacetyl-PEG2-Biotin, 50mg21901 EZ-Link Maleimide-PEG2-Biotin, 50mg29139 Biotinylated Horseradish Peroxidase, 5mgReferences1.Green, N.M. (1975). Avidin. In Adv. in Protein Chemistry. Academic Press, New York. 29:85-133.2.Green, N.M., et al. (1971). The use of bifunctional biotinyl compounds to determine the arrangement of subunits in avidin. Biochem. J. 125:781-91.3.Green, N.M. (1965). A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94:23c-24c.Products are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”). No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than Buyer. Any model or sample furnished to Buyer is merely illustrative of the general type and quality of goods and does not represent that any Product will conform to such model or sample.NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR ANY PARTICULAR PURPOSE, OR NON INFRINGEMENT. BUYER’S EXCLUSIVE REMEDY FOR NON-CONFORMING PRODUCTS DURING THE WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NON-CONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION. THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER, (III) USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED, OR (IV) IMPROPER STORAGE AND HANDLING OF THE PRODUCTS.Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to be used for any other purpose, including without limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption by or application to humans or animals.Current product instructions are available at /pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.© 2014 Thermo Fisher Scientific Inc. All rights reserved. Pronase is a trademark of Calbiochem. Unless otherwise indicated, all other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.。

生物素化免疫印迹法的原理生物素化免疫印迹法（Biotinylated Immunoblotting）是一种常用的蛋白质检测技术，广泛应用于生物医学研究和临床诊断中。

该方法利用生物素（Biotin）标记的抗体与特定蛋白质结合，再通过酶标记的亲生素（Streptavidin）结合生物素，通过酶催化反应使目标蛋白质产生可视化的信号。

本文将详细介绍生物素化免疫印迹法的原理及其应用。

生物素化免疫印迹法的原理基于特异性抗原与抗体的结合反应。

首先，待检样品经过蛋白质分离技术（如SDS-PAGE）将蛋白质分离并定位在聚丙烯酰胺凝胶上。

然后，将聚丙烯酰胺凝胶中的蛋白质转移到固体膜上，例如聚偏氟乙烯（PVDF）或硝酸纤维素膜。

这个过程称为电转印（Electrotransfer）。

接下来，将转印膜中的非特异性结合位点（例如未被蛋白质占据的空白位点）阻断，以防止后续步骤中的非特异性结合。

通常使用的阻断剂有非脂型乳清蛋白（非脂型BSA）或牛血清白蛋白（BSA）。

这样可以减少背景信号，提高检测的特异性。

然后，在转印膜上加入经生物素标记的一抗抗体，这种抗体能与待检测蛋白特异性结合。

生物素是一种小分子化合物，其分子量较小，与蛋白质结合不会影响其性质。

生物素标记的抗体与待检测蛋白特异性结合后，形成抗原-抗体复合物。

为了可视化待检测蛋白，将转印膜与酶标记的亲生素（通常是辣根过氧化物酶-HRP）结合。

亲生素是一种能与生物素非常紧密结合的蛋白质。

亲生素与生物素结合后，形成稳定的复合物。

然后，在转印膜上加入适当的底物，如4-氨基联苯二酚（4-chloro-1-naphthol），使HRP催化底物产生可视化的颜色反应。

产生的颜色与待检测蛋白的含量成正比。

生物素化免疫印迹法具有许多优点。

首先，该方法对待检测蛋白的特异性非常高，可以检测到非常低浓度的蛋白。

其次，由于生物素与亲生素结合稳定，可以进行长时间的信号增强，从而提高检测的灵敏度。

此外，生物素化免疫印迹法还可以同时检测多个蛋白，通过使用不同生物素标记的抗体，可以在同一转印膜上检测多个目标蛋白。

生物素-链霉卵白素免疫组化检测试剂盒使用说明Biotin-Streptavidin HRP Detection Systems规格:1kit保存:2-8℃避光保存产品简介：SP试剂盒，全称为过氧化物酶标记的链霉卵白素（Streptavidin/Peroxidase）检测试剂盒。

由于链霉卵白素的分子量是60kDa，有四个亚基可和生物素结合，等电点为6.5，所以该方法具有灵敏度高、特异性强、背景清晰、成本低廉的优点，但是由于使用了生物素，所以在某些内源性生物素含量比价高的组织中应慎用。

该系列检测试剂盒是性价比较高的免疫组化检测试剂。

试剂盒内容：试剂（无色液体）：3%H2O2去离子水3ml、6ml或18ml 试剂A（蓝色液体）：封闭用正常山羊血清工作液3ml、6ml或18ml 试剂B（黄色液体）：生物素标记山羊抗兔\小鼠IgG3ml、6ml或18ml 试剂C（橙色液体）：辣根酶标记链霉卵白素工作液（S-A/HRP）3ml、6ml或18ml 操作步骤:仅供参考1、石蜡切片，常规脱蜡至水。

2、如需采用抗原修复，在此步骤后进行。

3、3%H2O2去离子水（无色液体）孵育5~10分钟，以消除内源性过氧化物酶活性。

PBS 冲洗，3分钟×3次。

4、滴加试剂A（蓝色液体）室温孵育10~15分钟，倾去，勿洗。

5、滴加适当比例稀释的一抗，37℃孵育2~3小时或4℃过夜。

6、PBS冲洗，3分钟×3次。

7、滴加试剂B（黄色液体），室温或37℃孵育10~15分钟。

8、PBS冲洗，3分钟×3次。

9、滴加试剂C（橙色液体），室温或37℃孵育10~15分钟。

10、PBS冲洗，3分钟×3次。

11、显色剂显色（DAB或AEC）。

12、自来水充分冲洗。

13、如果需要，可进行复染，脱水，透明。

14、选择适当的封片剂封片。