蛋白磷酸酶抑制剂复合物I (100×)使用说明书
26146-Pierce
裂解/洗涤缓冲液（即 10:1 体积/质量） 。如果裂解大量细胞，可首先添加 10％终体积的 IP 裂解 /洗涤缓冲液到细胞团块中， 用移液管/移液器上下吹打细胞团块以混匀， 然后再向细胞悬浮液中 添加剩余的 IP 裂解/洗涤缓冲液。 4. 5. 将细胞裂解液在冰上孵育 5 分钟并定期混匀。〜 13000 × g 离心 10 分钟去除细胞碎片。 将上清转移到一个新的微量离心管中用于进行蛋白浓度测定和后续分析。
方案 II：裂解悬浮细胞培养物 1. 2. 3. 将细胞悬浮液在 1000 × g 下离心 5 分钟以沉淀细胞。弃掉上清。 将细胞团块用 PBS 重悬洗涤一次。1000×g 离心 5 分钟再次沉淀细胞。 将冰上预冷的 IP 裂解/洗涤缓冲液加到细胞团块中。每 50mg 湿重细胞团块加入 500 μL IP
3
掉流穿液体。 2. 用 100 μL 预冷的 IP 裂解/洗涤缓冲液洗涤树脂两次。每次洗涤后弃掉流穿液体。 3. 在纸巾上轻拍离心柱的底部以去除剩余的液体，插入底盖。 4. 将抗体/细胞裂解液样品加入含有蛋白 A/G 加强型琼脂糖的离心柱中。盖上螺旋盖温和地上 下翻转混匀或在摇床上震荡孵育 1 小时。 5. 卸下底盖，拧松螺旋帽并将离心柱置于收集管中。将柱子离心并保留流穿液体。在确定 IP 反应成功之前不要丢弃该液体。 6. 拧下螺旋帽，将柱子置于一个新的收集管中，加入 200 μL 的 IP 裂解/洗涤缓冲液并离心。 注：如果需要无去垢剂洗涤，则有另一款洗涤缓冲液（20×TBS 缓冲液）提供。使用前请 将该缓冲液稀释至 1×。 7. 用 200 μL IP 裂解/洗涤缓冲液洗涤树脂三次，并且每次洗涤后都进行离心。 8. 用 100 μL 1×条件缓冲液洗涤树脂一次。
储存：4℃储存，常温运输。
蛋白酶抑制剂
蛋白酶抑制剂引言蛋白酶抑制剂是一类用于抑制酶活性的化合物，其中蛋白酶是一种催化蛋白质水解的酶。
蛋白酶在生物体中起着重要的调控作用，参与多种生物反应的调节和控制。
然而，在某些情况下，蛋白酶的过度活化可能导致疾病的发生和发展。
因此，研发蛋白酶抑制剂成为一种重要的医药研究方向。
本文将介绍蛋白酶抑制剂的基本分类、作用机制以及在药物研发中的应用。
分类根据蛋白酶抑制剂对酶活性的抑制方式，蛋白酶抑制剂可分为两大类：1.反式抑制剂：反式抑制剂与酶结合后形成稳定的酶-抑制剂复合物，从而阻止酶催化反应的进行。
典型的例子是蛋白酶抑制剂Aprotinin，它通过与蛋白酶结合形成稳定的复合物，从而抑制蛋白酶的活性。
2.逆式抑制剂：逆式抑制剂通过与酶催化中心结合，改变酶的构象和活性。
这类抑制剂通常是模拟酶底物的分子结构，与酶催化中心发生特异性的相互作用。
逆式抑制剂的典型例子是酶的底物类似物，如硫氰酸酰胺（p-Tosyl-L-phenylalanine chloromethyl ketone，TPCK）可以选择性地抑制胰蛋白酶的活性。
作用机制蛋白酶抑制剂能够通过不同的机制来抑制蛋白酶的活性。
以下是几种常见的作用机制：1.受体拮抗作用：某些蛋白酶抑制剂能够与蛋白酶结合并阻断其与受体的相互作用，从而抑制了受体的活化。
这种机制在很多药物设计中都有重要的应用。
2.亲和力增强：一些蛋白酶抑制剂可以增强与酶的结合亲和力，从而阻止酶底物结合，进而抑制了酶的活性。
3.酶底物竞争：逆式抑制剂通过与酶催化中心竞争底物结合位点，从而阻断底物与酶的结合，进而抑制了酶活性的发生。
4.构象改变：某些蛋白酶抑制剂能够与酶发生特异性相互作用，改变酶的构象从而影响酶的活性。
应用蛋白酶抑制剂在医药领域有着广泛的应用。
以下是一些典型的应用示例：1.抗癌药物：蛋白酶在肿瘤细胞中起着重要的作用，因此蛋白酶抑制剂被广泛用于抗癌药物的研发。
例如，蛋白酶抑制剂Paclitaxel可以通过抑制肿瘤细胞内的蛋白酶活性来抑制细胞的增殖。
磷酸化-2011-硕士-课件(1)
研究发现 －→当Ca （二价 正离子）结合到E螺旋区和F螺 旋区之间的泡区时，引起每个
IV、cGMP依赖的蛋白激酶 (cGMP dependent protein kinase,,GPK)
Ashman 等1963年从肾脏首次发现cGMP
V、DNA依赖的蛋白激酶( DNA dependent protein kinase,DNA-PK)
是一类存在于细胞核内，能被DNA激活的特异的Ser/Thr PK 引起 多种核结合蛋白磷酸化。
蛋白激酶C为 77kd , 催化区抑制调节区,当DAG结合到蛋白激酶C上，解除酶的调节 区的抑制作用，使酶发挥催化活性。
III、Ca2+/钙调蛋白依赖的蛋白激酶
CaM（钙调蛋白）作为细胞内Ca2+受体，由148个氨基酸碱基组成的可溶性球蛋白 结构特点： （1 ）30%的酸性氨基酸，过量羧基提供了Ca2+可逆性结合的基团。 （2）不含能使肽链定型的成分，即易氧化的半胱氨酸和羟脯氨酸，因而CaM具有 高度灵活的，可与机体蛋白相互作用的三级结构。
（3）与蛋白/肽底物竞争抑制剂 Walsh：是较为特异的APK抑制剂。同肽底物竞争
（三）蛋白磷酸酯酶（protein phosphatase 。） 70年代末PTPase活性首次被发现，已知对Ser/Thr激酶的磷酸酯酶有 PP1,PP2A,PP2B,PP2C,PPx等分布涉及各组织以至细胞器，定位各有有 侧重，均有亚型 PP1(位于胞浆为为PP11 糖原分子为PP1G,位于肌丝为PP1M,核为PP1N) PP2A主要存在胞浆，少数在线粒体和核 PP2B与膜连接有关（调节亚单位 氨基末端甘氨酸残基豆蔻化） PP2C只要存在于胞浆 PPC存在于细胞膜 PPx存在于线粒体
β-Glycerophosphate (sodium salt hydrate)_蛋白磷酸酶抑制剂_13408-09-8_Apexbio
donor in bone cell mineralization studies [1][3]. In MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells, β-Glycerophosphate did not affect the rate of anaerobic glycolysis, but increased the medium Pi levels, suggesting that β-Glycerophosphate was hydrolyzed by bone cells. The local increase in medium Pi concentration promoted rapid mineral deposition [1]. In human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB), BMP-2 resulted in mineralization of their matrix in the presence of beta-glycerophosphate and ascorbic acid [3]. β-Glycerophosphate (10 mM) also accelerated calcification of cultured vascular smooth muscle cells through an alkaline phosphatase-related mechanism [4]. 参考文献:
产品描述:
β-Glycerophosphate (sodium salt hydrate) is a potent protein phosphatase inhibitor [1]. Protein phosphatase is an enzyme that removes a phosphate group from the phosphorylated amino acid residue of its target protein. Protein phosphorylation is one of the most common protein posttranslational modifications. β-Glycerophosphate is a potent protein phosphatase inhibitor that acts as a phosphate group
蛋白质提取过程中常用的蛋白酶和磷酸酶抑制剂详细使用说明
蛋白质提取过程中常用的蛋白酶和磷酸酶抑制剂详细使用说明转自:http://www.bioas/html/980.html在与蛋白相关的检测中，最关键的一步便是蛋白质的提取。
在提取的过程中，我们要经常加入以防止。
另外在磷酸化蛋白的研究过程中，也是必不可少的。
本文详细总结了常用的P MSF、 Leupep tin亮肽素、Aproti nin抑肽酶、Pepsta tin胃、EDTA-Na2等以及NaF氟化钠、Na3VO4原矾酸钠、Beta-glycer ophos phate甘油磷酸钠、Na2P2O4焦磷酸钠等的溶液配制、贮存液与工作液浓度及保存条件。
一、蛋白酶抑制剂PMSF：特性：丝氨酸蛋白酶抑制剂，如胰凝乳蛋白酶、胰蛋白酶和凝血酶，也抑制半胱氨酸蛋白酶如木瓜蛋白酶。
溶解性：溶于异丙醇、乙醇、甲醇和丙二醇里>10mg/ml。
在水溶液中不稳定。
在100%异丙醇，25℃时稳定至少9个月。
分子量：174.2使用：贮存浓度200mM，工作浓度1m MLeupep tin 亮肽素特性：抑制丝氨酸和半胱氨酸蛋白酶如胰蛋白酶、木瓜蛋白酶、纤溶酶和组织蛋白酶B。
溶解性：高度溶于水（1mg/ml）。
4℃一周稳定，分成小份，冷冻在 -20℃至少6个月。
分子量：C20H38N6O4×1/2 H2SO4:475.6 C20H38N6O4x 1/2 H2SO4× H2O:493.6使用：贮存浓度1m g/ml，工作浓度0.5 ug/ml (1mM)。
Aproti nin抑肽酶特性：丝氨酸蛋白酶抑制剂，抑制纤维蛋白溶酶、激肽释放酶、胰蛋白酶、糜蛋白酶的高活性。
不抑制凝血酶或因子X。
溶解性：易溶于水（10mg/ml）或缓冲液（例如0.1M tris，pH8.0）。
蛋白酶和磷酸酶抑制剂详细使用说明
蛋白质提取过程中常用的蛋白酶和磷酸酶抑制剂详细使用说明一、蛋白酶抑制剂PMSF：特性：丝氨酸蛋白酶抑制剂，如胰凝乳蛋白酶、胰蛋白酶和凝血酶，也抑制半胱氨酸蛋白酶如木瓜蛋白酶。
溶解性：溶于异丙醇、乙醇、甲醇和丙二醇里>10mg/ml。
在水溶液中不稳定。
在100%异丙醇，25℃时稳定至少9个月。
分子量：174.2使用：贮存浓度200mM，工作浓度1mMLeupeptin 亮肽素特性：抑制丝氨酸和半胱氨酸蛋白酶如胰蛋白酶、木瓜蛋白酶、纤溶酶和组织蛋白酶B。
溶解性：高度溶于水（1mg/ml）。
4℃一周稳定，分成小份，冷冻在-20℃至少6个月。
分子量：C20H38N6O4 ×1/2 H2SO4:475.6 C20H38N6O4 x 1/2 H2SO4 × H2O:493.6使用：贮存浓度1mg/ml，工作浓度0.5 ug/ml (1mM)。
Aprotinin抑肽酶特性：丝氨酸蛋白酶抑制剂，抑制纤维蛋白溶酶、激肽释放酶、胰蛋白酶、糜蛋白酶的高活性。
不抑制凝血酶或因子X。
溶解性：易溶于水（10mg/ml）或缓冲液（例如0.1M tris，pH8.0）。
pH约7~8的溶液在4℃可保存1周，分装保存在-20℃可至少保存6个月。
避免反复冻融, pH>12.8的碱性环境可使其灭活。
分子量：6,512使用：贮存浓度1mg/ml，工作浓度0.06~2.0 ug/ml(0.01~0.3 uM)。
Pepstatin胃蛋白酶抑制剂特性：抑制天冬氨酸（酸）蛋白酶如胃蛋白酶、肾素、组织蛋白酶D、凝乳酶、许多微生物酸性蛋白酶溶解性：溶于甲醇约1mg/ml；可溶于乙醇，过夜溶解可达到1 mg/ml；在6当量乙酸中溶解度为300ug/ml。
4℃稳定一周，分装储存于-20℃时可保存1个月。
分子量：685.9使用：贮存浓度1mg/ml，使用浓度0.7 μg/ml(1 μM)。
EDTA-Na2特性：金属蛋白酶抑制剂溶解性：溶于水至0.5M，在pH8-9的条件下，4℃稳定至少6个月分子量：372.24使用：工作浓度0.2~0.5 mg/ml(0.5~1.3 mM)，不需现用现配，在溶液pH值调至8~9时再加入。
免疫沉淀(Immunoprecipitation)实验操作方法
免疫沉淀（Immunoprecipitation）实验操作方法实验原理：免疫沉淀（Immunoprecipitation，IP）是利用抗原和抗体的特异性结合以及细菌蛋白质如protein A/G特异性地结合到免疫球蛋白（抗体）FC片段的现象而开发出来的特异分离富集抗原的方法。
目前多用预先结合固化在Agarose beads上的protein A/G（Agrose-proteinA/G），使之与抗体结合，再通过抗体特异性结合抗原而形成Agrose-proteinA/G 、抗体、抗原复合物，利用Agarose beads的重量，通过离心即可特异分离富集目的抗原。
实验步骤：1.处理细胞：依据实验目的，设计实验，处理好细胞模型。
一般3×106细胞/处理(即六孔板一个孔，约200ug总蛋白)。
2.配IP 裂解液：200ul/孔（6孔板），并加入蛋白酶抑制剂，如果蛋白磷酸化水平影响实验结果则应加入磷酸酶抑制剂。
注意抑制剂的要现加现用。
3.收集细胞：冰上操作，裂解细胞前用1×PBS（4度）洗1遍，每孔加入200ul IP裂解液，冰上静置5分钟后刮下细胞并收集至EP管中。
4.超声：强度37％，5秒×4次（程序07）。
目的是使细胞裂解更完全并打断基因组DNA,但可能会影响蛋白质之间的相互作用，进行共免疫沉淀实验时要注意这一点。
5.离心细胞裂解液：4度10000rpm 10 分钟。
6.Agrose-proteinA/G清洗：一般1ug抗体对应15ul Agrose-proteinA/G（不同公司产品可能不同，注意结合说明书及实验结果考虑），预清洗细胞裂解液的Agrose-proteinA/G用量与结合抗体的用量一致。
取相应量的Agrose-proteinA/G到EP管中（剪枪头）并用500ul1×PBS（4度）洗两遍，5000rpm×2分钟，吸掉上清备用。
7.细胞裂解液预清洗：为去除非特异作用，细胞裂解液先用Agrose-proteinA/G预清洗。
S100蛋白(S100)作业指导书
检验科免疫室分析项目作业指导书第页，共页版本：A/0生效日期：2008-02-01 36b)>95%健康成人范围的样本量c)没有疾病，不患肿瘤可能为颅脑损伤的成年患者表现为轻微颅脑外伤（格拉斯哥昏迷计分GCS：13-15分）和至少有一种症状的患者3小时内进行Elecsys S100测定。
外伤后6小时内必须进行CCT（头颅计算机X线断层摄影术）检查。
以健康个体的95%值作为分界点，CCT作为参照，用Elecsys S100测定可获得如下结果：NPV（阴性预测值）99.7%，PPV（阳性预测值）11%，灵敏度98.8%，和特异度32.9%（95%可信区间：NPV99.1-100%，PPV8.8-13.3%,灵敏度96.5-100%，特异度30-35.9%）。
d)0.098μg/L每个实验室必须调查各自患者群体的参考范围变异性，必要时根据具体情况制订自己的参考范围。
9.分析性能分析仪的代表性试验数据如下。
各实验室由于具体情况不同，所得出的数据可能会稍有差异。
精密度根据NCCLS（全国临床实验室规范化操作委员会）制订的改良试验计划(EP5-A)，应用Elecsys试剂盒、人血液标本和质控液验证ElecsysS100试剂盒检测重复性。
每日测6 次共10 日（n＝60）；在E170 分析仪上的组间精密度（n＝21），得结果如下：分析灵敏度（低浓度检测限制）< 0.005 µg/L检验科免疫室分析项目作业指导书第页，共页版本：A/0生效日期：2008-02-01 46检测限制定义为可以区别零浓度定标液的最低可测浓度值，计算公式为定标液最小浓度＋2SD（标准差）（系统定标，定标液最小浓度＋2SD，组间精密度范围之内，标本数21）。
方法比较将Elecsys S100 现在的试剂盒Elecsys S100（产品目录号03051986（y））与Liamat Sangtec100 (x1)和 Liaison Sangtec100 (x2)进行比较，得到以下线性关系(pmol/L)。
蛋白酶抑制剂混合物(哺乳动物样品抽提用, 100X)
北京索莱宝科技有限公司蛋白酶抑制剂混合物(哺乳动物样品抽提用,100X)货号：P6731规格：100T保存：-20ºC保存，有效期12个月；4ºC保存，2个月有效；室温保存2周有效。
产品组成：产品名称规格保存蛋白酶抑制剂混合物(哺乳动物样品抽提用,100X)1mL-20ºC避光0.5M EDTA,pH8.01mL RT产品说明：哺乳动物细胞或组织提取物中含有许多内源性的蛋白酶、磷酸酶等，容易导致提取物中的蛋白降解或去修饰，从而影响后续的蛋白检测。
因此在提取物中添加适当的蛋白酶、磷酸酶等抑制剂是防止蛋白降解和去修饰的有效方法。
本产品用于哺乳动物细胞或组织蛋白提取的蛋白酶抑制剂混合物，包含了广谱的丝氨酸、半胱氨酸和酸性蛋白酶抑制剂、以及氨基肽酶抑制剂。
如用于检测金属蛋白酶活性，则不宜添加EDTA。
以1:100的比例分别把蛋白酶抑制剂混合物(哺乳动物样品抽提用,100X)和0.5M EDTA加入裂解液中，即可用于哺乳动物细胞或组织蛋白的提取，并有效抑制蛋白降解。
适用范围：抑制哺乳动物细胞或组织提取物中的各种蛋白酶活性，如丝氨酸蛋白酶、氨基肽酶、半胱氨酸蛋白酶、苏氨酸和天冬氨酸蛋白酶、金属蛋白酶等。
适用于Western Blot和免疫共沉淀检测磷酸化蛋白质、蛋白激酶活性测定等。
使用方法：1、蛋白酶抑制剂混合物（100X），使用时分别按照1:100的比例加入到裂解液中，混匀后即可使用。
含有蛋白酶抑制剂混合物的裂解液宜现用现配，不宜配制后冻存待后续使用。
2、根据需要，0.5M的EDTA也按照1:100的比例加入到裂解液中。
第1页共1页。
人S100钙结合蛋白A8 A9复合物(S100A8 A9)Elisa kit实验操作说明书中英文
Human S100 Calcium-Binding Protein A8/A9 Complex (S100A8/A9) ELISA KitCatalog Number. CSB-E12149hFor the quantitative determination of human S100calcium-binding protein A8/A9complex(S100A8/A9)concentrations in serum,plasma, tissue homogenates.This package insert must be read in its entirety before using this product.In order to obtain higher efficiency service, please ready to supply the lot number of the kit to us (found on the outside of the box).PRINCIPLE OF THE ASSAYThis assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for S100A8/A9has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any S100A8/A9 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for S100A8/A9 is added to the wells. After washing,avidin conjugated Horseradish Peroxidase(HRP) is added to the wells.Following a wash to remove any unbound avidin-enzyme reagent,a substrate solution is added to the wells and color develops in proportion to the amount of S100A8/A9bound in the initial step.The color development is stopped and the intensity of the color is measured.D ETECTION RANGE1.56 ng/ml-100 ng/ml.SENSITIVITYThe minimum detectable dose of human S100A8/A9 is typically less than 0.39 ng/ml.The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean O.D value of 20 replicates of the zero standard added bytheir three standard deviations.SPECIFICITYThis assay has high sensitivity and excellent specificity for detection of human S100A8/A9.No significant cross-reactivity or interference between human S100A8/A9 and analogues was observed.Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between human S100A8/A9 and all the analogues, therefore, cross reaction may still exist.PRECISIONIntra-assay Precision (Precision within an assay): CV%<8%Three samples of known concentration were tested twenty times on one plate to assess.Inter-assay Precision (Precision between assays):CV%<10%Three samples of known concentration were tested in twenty assays to assess.LIMITATIONS OF THE PROCEDUREFOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time or temperature,and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay,the possibility of interference cannot be excluded.MATERIALS PROVIDEDSTORAGE*Provided this is within the expiration date of the kit.OTHER SUPPLIES REQUIREDMicroplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.An incubator which can provide stable incubation conditions up to 37°C±0.5°C.Squirt bottle, manifold dispenser, or automated microplate washer.Absorbent paper for blotting the microtiter plate.100ml and 500ml graduated cylinders.Deionized or distilled water.Pipettes and pipette tips.Test tubes for dilution.PRECAUTIONSThe Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.SAMPLE COLLECTION AND STORAGESerum Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 15 minutes at 1000 ×g. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.Plasma Collect plasma using EDTA, or heparin as an anticoagulant.Centrifuge for 15minutes at1000x g,2 - 8°C within30minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay.Tissue Homogenates100mg tissue was rinsed with1X PBS, homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes,the homogenates were centrifuged for 5 minutes at 5000 x g, 2 - 8°C. The supernate was removed and assayed immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.Note:1.CUSABIO is only responsible for the kit itself, but not for the samplesconsumed during the assay.The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.2.Samples to be used within 5 days may be stored at 2-8°C, otherwisesamples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid loss of bioactivity and contamination.3.Grossly hemolyzed samples are not suitable for use in this assay.4.If the samples are not indicated in the manual, a preliminary experimentto determine the validity of the kit is necessary.5.Please predict the concentration before assaying. If values for these arenot within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.6.Tissue or cell extraction samples prepared by chemical lysis buffer maycause unexpected ELISA results due to the impacts of certain chemicals.7.Owing to the possibility of mismatching between antigen from otherresource and antibody used in our kits(e.g.,antibody targets conformational epitope rather than linear epitope),some native or recombinant proteins from other manufacturers may not be recognized by our products.8.Influenced by the factors including cell viability, cell number and alsosampling time, samples from cell culture supernatant may not be detected by the kit.9.Fresh samples without long time storage are recommended for the test.Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.REAGENT PREPARATIONNote:Kindly use graduated containers to prepare the reagent. Please don't prepare the reagent directly in the Diluent vials provided in the kit.Bring all reagents to room temperature (18-25°C) before use for 30min.Prepare fresh standard for each assay. Use within 4 hours and discard after use.Making serial dilution in the wells directly is not permitted.Please carefully reconstitute Standards according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved.To minimize imprecision caused by pipetting,use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result.1.Biotin-antibody (1x) - Centrifuge the vial before opening.Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μl of Biotin-antibody + 990 μl of Biotin-antibody Diluent.2.HRP-avidin (1x) - Centrifuge the vial before opening.HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10μl of HRP-avidin + 990 μl of HRP-avidin Diluent.3.Wash Buffer (1x) - If crystals have formed in the concentrate, warm up toroom temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 ml of Wash Buffer (1 x).4.StandardCentrifuge the standard vial at 6000-10000rpm for 30s.Reconstitute the Standard with 1.0ml of Sample Diluent.Do not substitute other diluents. This reconstitution produces a stock solution of 100 ng/ml. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.Pipette 250 μl of Sample Diluent into each tube (S0-S6). Use the stock solution to produce a2-fold dilution series(below).Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard (100 ng/ml). Sample Diluent serves as the zero standard (0 ng/ml).ASSAY PROCEDUREBring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay.It is recommended that all samples and standards be assayed in duplicate.1.Prepare all reagents, working standards, and samples as directed in theprevious sections.2.Refer to the Assay Layout Sheet to determine the number of wells to beused and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.3.Add 100μl of standard and sample per well. Cover with the adhesive stripprovided.Incubate for2hours at37°C. A plate layout is provided to record standards and samples assayed.4.Remove the liquid of each well, don’t wash.5.Add100μl of Biotin-antibody(1x)to each well.Cover with a newadhesive strip. Incubate for 1 hour at 37°C. (Biotin-antibody(1x)may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)6.Aspirate each well and wash, repeating the process two times for a totalof three washes. Wash by filling each well with Wash Buffer (200μl) usinga squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,and let it stand for 2 minutes, complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.7.Add 100μl of HRP-avidin(1x) to each well. Cover the microtiter plate witha new adhesive strip. Incubate for 1 hour at 37°C.8.Repeat the aspiration/wash process for five times as in step 6.9.Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at37°C. Protect from light.10.Add 50μl of Stop Solution to each well, gently tap the plate to ensurethorough mixing.11.Determine the optical density of each well within5minutes,using amicroplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.Note:1.The final experimental results will be closely related to validity of theproducts,operation skills of the end users and the experimental environments.2.Samples or reagents addition: Please use the freshly prepared Standard.Please carefully add samples to wells and mix gently to avoid foaming.Do not touch the well wall as possible. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.3.Incubation: To ensure accurate results, proper adhesion of plate sealersduring incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps.Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be observed.4.Washing: The wash procedure is critical. Complete removal of liquid ateach step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate.Insufficient washing will result in poor precision and falsely elevated absorbance reading. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate180 degrees between wash steps may improve assay precision.5.Controlling of reaction time: Observe the change of color after addingTMB Substrate (e.g. observation once every 10 minutes), TMB Substrate should change from colorless or light blue to gradations of blue. If the color is too deep,add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.6.TMB Substrate is easily contaminated.TMB Substrate should remaincolorless or light blue until added to the plate. Please protect it from light.7.Stop Solution should be added to the plate in the same order as the TMBSubstrate. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the TMB Substrate.ASSAY PROCEDURE SUMMARY*Please determine whether the sample needs to be diluted or the optimal dilution factor based on preliminary experiment result.CALCULATION OF RESULTSUsing the professional soft "Curve Expert" to make a standard curve is recommended, which can be downloaded from our web.Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic(4-PL)curve-fit.As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the S100A8/A9 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis.This procedure will produce an adequate but less precise fit of the data.If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.人S100钙结合蛋白A8/A9复合物(S100A8/A9)酶联免疫试剂盒使用说明书【产品编号】CSB-E12149h【预期应用】ELISA法定量测定人血清、血浆、组织裂解液中S100A8/A9含量。
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
使 用 说 明 书
◆蛋白磷酸酶抑制剂复合物I (100×)
◆目录号1913
◆使用手册
◆实验室使用，仅用于体外
蛋白磷酸酶抑制剂复合物I (100×)
目录号：1913
目录编号包装单位
191301 1 ml
191302 1 ml× 5
适用范围：
抑制蛋白丝/苏氨酸磷酸酶、蛋白酪氨酸磷酸酶、碱性磷酸酶、酸性磷酸酶
产品储存：
－20℃保存，一年有效。
产品介绍：
组织/细胞裂解时会释放出大量内源性蛋白磷酸酶，以各种方式催化磷酸化蛋白的去磷酸化，导致不同于正常生理状态的差异。
因此，裂解后的Western blotting，免疫共沉淀检测磷酸化蛋白质、和蛋白激酶活性测定等实验常规要加入外源性蛋白磷酸酶抑制剂，抑制磷酸化蛋白的去磷酸化，维持蛋白质的磷酸化状态。
本公司研制的蛋白磷酸酶抑制剂复合物以国际上主流配方改进而成，主要成份为5种独立的蛋白磷酸酶抑制剂（钒酸钠、氟化钠、钼酸钠、酒石酸钠、咪唑），每一种抑制剂可特异性抑制某一种或几种蛋白磷酸酶活性。
该复合物优化的组成使其可以强烈抑制几乎所有重要的蛋白磷酸酶活性，包括蛋白丝/苏氨酸磷酸酶、蛋白酪氨酸磷酸酶、碱性磷酸酶、酸性磷酸酶以及ATP酶等。
该复合物为100倍浓缩溶液，可直接加入任何组织类型和细胞的裂解产物中，均能有效抑制磷酸化蛋白质的去磷酸化，从而维护蛋白质的磷酸化状态。
适用于Western blot和免疫共沉淀检测磷酸化蛋白质、蛋白激酶活性测定等。
操作步骤：
临用前室温溶解磷酸酶抑制剂复合物I (100×)，混匀，按照1：100比例加入到组织细胞裂解物中。
提示：
⇨避免反复冻融，第一次使用后，可按照每次使用量分装冻存。
临用前加入，终浓度为1×，蛋白磷酸酶含量特别丰富的组织，可以用2-3×。
完。