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细胞相关> 细胞死亡与自噬> 细胞凋亡
Caspase 3 活性检测试剂盒
产品编号: C1116
产品包装:100次
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价格:¥1780.00元
产品简介
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产品编号 产品名称 产品包装 产品价格
C1115 Caspase 3 活性检测试剂盒 20次 512.00元
C1116 Caspase 3 活性检测试剂盒 100次 1780.00元

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caspase 3,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caspaseCaspase 3可以剪切procaspase 3、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 condensation),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。
Caspase 3活性检测试剂盒是基于caspase 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。pNA405nm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100μl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:

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) 200μl/管,共5管
C1116-4 pNA(10mM) 1ml
说明书 1份

保存条件:
-20℃保存,Ac-DEVD-pNA和pNA需避光保存。
注意事项:
须自备可以测定A405或A400的酶标仪或容量不超过100μl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-YVAD-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)对人体有毒,操作时请特别小心,并注意有效防护以避免直接接触人体或吸入体内。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。
本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
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:
1.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pNA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200μM,作为标准品。
c.每个浓度取100μl用酶标仪进行检测,或取适当量用容量不超过 100μl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浓度相对于A405的标准曲线。pNA标准曲线可以参考图 1,在0-200μM范围内存在良好的线性关系。


1.pNA标准曲线。实测数据可能因实验条件、检测仪器等的不同而存在差异,图中数据仅供参考。

3.样品的收集:
a.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(如果裂解不充分,可以把裂解液的用量提高至150或200微升),重悬沉淀,冰浴裂解15分钟。下转步骤3d
b.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(如果裂解不充分,可以把裂解液的用量提高至150或200微升),重悬沉淀,冰浴裂解15分钟。下转步骤3d
c.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微升待测样品中至少含有10-30μg蛋白。如果细胞较小,可以适当增加细胞的用量。
4.Caspase 3酶活性的检测:
a.取出适量的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 40μl 40μl
待测样品 0μl 50μl
裂解液 50μl 0μl
Ac-DEVD-pNA (2mM) 10μl 10μl
总体积 100μl 100μl

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10μl 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 thatwill cleave1.0nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37℃ undersaturated 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- 产生pNA1nmol 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: 
1. 测定出的A405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至少达到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根据具体情况而定。 
2. 测定出的A405过高或者样品量不足: 
测定出来的A405读数过高时,可以参考下表的反应体系适当减少样品的用量;样品量不足时也可以参考下表减少样品 的用量。

  空白对照 样品
检测缓冲液 40μl 40μl
待测样品 0μl xμl
裂解液 50μl (50-x)μl
Ac-DEVD-pNA (2mM) 10μl 10μl
总体积 100μl 100μl

说明:其中x不超过50,其余检测方法同上面的使用说明所述。

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