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Caspase 8 活性检测试剂盒
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产品编号: C1152
产品包装: 100次
产品价格: 1758.00元 |
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产品简介
产品简介:
Caspase 8 活性检测试剂盒(Caspase 8 Activity Assay Kit)是采用分光光度法检测细胞或组织裂
解液中caspase 8酶活性或纯化的caspase 8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 8也称FLICE、MACH或Mch5,有时被写作caspase-8或caspase 8,通常以酶原的形式存在,在细胞凋亡的信号转导过程中被激活。Caspase 8被认为是细胞凋亡信号转导过程中比较上游的一个caspase。在Fas-receptor和TNFR-1介导的细胞凋亡过程中caspase 8被激活,形成一个由p18和p10组成的二聚体,进一步激活下游的caspase 4,caspase 6,caspase 9和caspase 10。
本Caspase 8活性检测试剂盒是基于caspase 8可以催化底物Ac-IETD-pNA(acetyl-Ile-Glu-Thr-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 8的活性。pNA在405nm附近有强吸收。
试剂盒中提供了caspase 8催化产生的黄色产物pNA,可以作为定量caspase 8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 |
C1152-1 |
裂解液 |
30ml |
C1152-2 |
检测缓冲液 |
10ml/瓶,共2瓶 |
C1152-3 |
Ac-IETD-pNA(2mM) |
200μl/管,共5管 |
C1152-4 |
pNA(10mM) |
1ml |
— |
说明书 |
1份 |
保存条件:
-20℃保存,Ac-IETD-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-IETD-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。
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:
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。
图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,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,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 8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 8酶活性的检测:
a.取出适量的Ac-IETD-pNA(2mM),置于冰浴上备用。
b.如下设置反应体系: |
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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-IETD-pNA(2mM) |
10μl |
10μl |
总体积 |
100μl |
100μl |
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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-IETD-pNA(2mM)。
c.加入Ac-IETD-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 8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 8酶活力单位的定义:One unit is the amount of enzyme that
will cleave 1.0nmol of the colorimetric substrate Ac-IETD-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-IETD-pNA产生1nmol pNA的caspase 8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 8。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 8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 8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。 |
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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-IETD-pNA(2mM) |
10μl |
10μl |
总体积 |
100μl |
100μl |
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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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