C1116 Caspase 3 活性检测试剂盒.pdf

Caspase 3 活性检测试剂盒活性检测试剂盒 产品编号 产品名称 包装 C1116 Caspase 3 活性检测试剂盒 100次 产品简介：产品简介：Caspase 3 活性检测试剂盒(Caspase 3 Activity Assay Kit)是采用分光光度法检测细胞或组织裂解液中Caspase 3酶活性或纯化的Caspase 3酶活性的试剂盒。Caspase(Cysteine-requiring Aspartate Protease)是一个在细胞凋亡过程中起重要作用的蛋白酶家族。Caspase 3也称CPP32、Yama或apopain，有时被写作caspase-3或caspase3，属于caspase家族的CED-3亚家族(CED-3 subfamily)，是细胞凋亡过程中的一个关键酶。Caspase 3是哺乳动物细胞中研究最多的一个caspase。Caspase 3可以剪切procaspase 2、6、7和9，并可以直接 特 异 性 剪 切 许 多 caspase 底 物，包 括 PARP(poly(ADP-ribose)polymerase)，ICAD(Inhibitor of caspase-activated deoxyribonuclease)，gelsolin和fodrin等。这些由caspase 3介导的蛋白剪切是细胞凋亡分子机制的重要组成部分。另外，caspase 3在细胞核凋亡过程中也起到了关键作用，包括染色质固缩(chromatin condensention)，DNA片段化(DNA fragmentation)等。同时caspase 3对细胞起泡(Cell blebbing)也起到关键作用。本Caspase 3 活性检测试剂盒是基于casepase 3可以催化底物Ac-DEVD-pNA(acetyl-Asp-Glu-Val-Asp p-nitroanilide)产生黄色的pNA(p-nitroaniline)，从而可以通过测定吸光度来检测caspase 3的活性。pNA在405nm附近有强吸收。试剂盒中提供了caspase 3催化产生的黄色产物pNA，可以作为定量caspase 3酶活性的标准品。本试剂盒用酶标仪检测或容量不超过100l的分光光度检测杯检测时，除标准曲线外可以检测100个样品。包装清单：包装清单：产品编号 产品名称 包装 C1116-1 裂解液 30ml C1116-2 检测缓冲液 10ml/瓶，共2瓶 C1116-3 Ac-DEVD-pNA(2mM)200l/管，共5管 C1116-4 pNA(10mM)1ml 说明书 1份 保存条件：保存条件：-20保存，Ac-DEVD-pNA和pNA需避光保存。注意事项：注意事项：须自备可以测定A405或A400的酶标仪或容量不超过100l的分光光度检测杯及相应分光光度计。优先考虑测定A405，如有困难可以测定A400。Ac-DEVD-pNA需尽量避免反复冻融，请注意适当分装。测定蛋白浓度需Bradford蛋白浓度测定试剂盒(P0006)，可向碧云天订购。建议样品用水稀释1倍后再用Bradford法测定蛋白浓度，以降低DTT 对蛋白浓度测定的干扰。有文献报道少数类型的细胞凋亡检测不到caspase 3的激活。pNA(中文名为4-硝基苯胺)有毒，请注意小心防护。pNA(10mM)在4、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25水浴温育片刻至全部融解后使用。本试剂盒的裂解液可以和碧云天生产的其它Caspase活性检测试剂盒的裂解液通用，即本试剂盒裂解液制备的蛋白样品可以用于碧云天其它Caspase活性检测试剂盒的检测。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明：使用说明：1.准备工作：准备工作：A.裂解液溶解后混匀并置于冰浴上备用。B.检测缓冲液溶解后混匀并置于冰浴上备用。2.测定测定pNA标准曲线标准曲线：A.标准品稀释液的配制：按照每0.9ml检测缓冲液加入0.1ml裂解液的比例配制适量的标准品稀释液。B.把试剂盒提供的pNA(10mM)用标准品稀释液稀释为0、10、20、50、100和200M，作为标准品。C.每个浓度取100l用酶标仪进行检测，或取适当量用容量不超过100l的分光光度检测杯进行检测，测定A405。D.每一个标准品的A405减去不含pNA的空白对照的A405计算出实际的因pNA而导致的吸光度，并制作出pNA浓度相当于A405的标准曲线。3.样品的收集样品的收集：A.对于悬浮细胞：对于悬浮细胞：把没有诱导凋亡的对照样品和诱导凋亡的样品，600g 4离心5分钟收集细胞，小心吸除上清，同时确保尽量没有细胞被吸除，PBS洗涤一次。同前吸尽上清后，按照每200万细胞加入100微升裂解液的比例加入裂解液，重悬沉淀，冰浴裂解15分钟。下转步骤3D。B.对于贴壁细胞：对于贴壁细胞：吸取细胞培养液，备用。用胰酶消化贴壁细胞，并收集至备用的细胞培养液中。600g 4离心5分钟收集细胞，小心吸除上清，同时确保尽量没有细胞被吸除，PBS洗涤一次。同前吸尽上清后，按照每200万细胞加入100微升裂解液的比例加入裂解液，重悬沉淀，冰浴裂解15分钟。下转步骤3D。C.对于组织样品：对于组织样品：按照每3-10mg组织加入100微升裂解液的比例加入裂解液，在冰浴上用玻璃匀浆器匀浆。然后把匀浆液转移到1.5ml离心管中，冰浴再裂解5分钟。D.4 16,000-20,000g离心10-15分钟。E.把上清转移到冰浴预冷的离心管中。F.立即测定caspase 3的酶活性或-70保存样品。同时可以取少量样品用Bradford法测定蛋白浓度，尽量使蛋白浓度达到1-3mg/ml，相当于每10微升待测样品中含有10-30g蛋白。如果细胞较小，可以适当增加细胞的用量。4.Caspase 3酶活性的酶活性的检测：检测：A.取出pNA和适量的Ac-DEVD-pNA(2mM)，置于冰浴上备用。B.如下设置反应体系：空白对照 样品 检测缓冲液 90l 80l 待测样品 0l 10l Ac-DEVD-pNA(2mM)10l 10l 总体积 100l 100l 注意：注意：在设置反应体系时先加检测缓冲液，再加待测样品，适当混匀，注意避免在混匀时产生气泡。随后再加入10l Ac-DEVD-pNA(2mM)。C.加入Ac-DEVD-pNA(2mM)后混匀，注意避免在混匀时产生气泡。37孵育60-120分钟。发现颜色变化比较明显时即可测定A405。如果颜色变化不明显，可以适当延长孵育时间，甚至可以孵育过夜。D.样品的A405扣除空白对照的A405，即为样品中caspase 3催化产生的pNA产生的吸光度。通过同步骤1中获得的标准曲线的对比就可以计算出样品中催化产生了多少量的pNA。E.参考Chemicon公司的caspase 3酶活力单位的定义：One unit is the amount of enzyme that will cleave 1.0 nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37 under saturated substrate concentrations。即一个酶活力单位定义为当底物饱和时，在37一个小时内可以剪切1nmol Ac-DEVD-pNA产生1nmol pNA的caspase 3的酶量。这样就可以计算出样品中含有多少个酶活力单位的caspase 3。说明：在本试剂盒的检测体系中，底物的起始浓度为0.2mM，此时底物是饱和的，对于许多样品而言在37孵育2个小时以内底物都是饱和的；对于样品中caspase 3酶活力特别高的情况，须用裂解液适当稀释样品后再进行测定。F.用Bradford法检测待测样品中的蛋白浓度(由于裂解液中含有较高浓度的DTT，不适合采用BCA法进行蛋白浓度测定)。这样就可以计算出一个样品单位重量蛋白中所含的caspase 3的酶活力单位。常见问题常见问题：1.测定出的测定出的A405过低过低：A.样品中蛋白含量太低，裂解样品时需设法使样品中的蛋白浓度接近1-3mg/ml。B.样品中激活的caspase水平很低。首先确认凋亡现象是否明显，如果凋亡比较明显并且确认该caspase是可以被激活的，可以适当调节诱导细胞凋亡的时间，希望能找到一个caspase激活比较强的时间点，这样就可以检测出该caspase的激活。可以作一时间曲线，例如诱导凋亡0、2、4、8、16和24小时，或0、1、2、4、8和16小时，或0、1、2、4、6和8小时等。具体的诱导凋亡时间需更据具体情况而定。C.通过上述优化后A405还是比较低，或浓缩样品或做时间曲线比较困难，可以在测定样品时加大样品的用量，最多可达40l。假设每次测定样品的用量为xl，则：C1.此时标准品稀释液需按如下方法配制：按照xl裂解液加入检测缓冲液至最终体积为100l的比例配制适量的标准品稀释液。例如样品用量为40l，则按照40l裂解液加入60l检测缓冲液，即0.4ml裂解液加入0.6ml检测缓冲液的比例配制标准品稀释液。其余标准曲线的测定方法同上面的使用说明所述。C2.此时如下设置反应体系：空白对照 样品 检测缓冲液 90l(90-x)l 待测样品 0l xl Ac-DEVD-pNA(2mM)10l 10l 总体积 100l 100l 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