Caspase-3 Activity Assay Kit

• 产品编号：CST-5723S      品牌：CST       原厂货号：5723S
• 产品分类：生化和细胞分析 > 细胞分析试剂盒 > 细胞凋亡试剂盒
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描述：

Caspase-3活性检测试剂盒检测凋亡细胞中活化的caspase-3裂解的AC-DEVD-AMC产生的AMC荧光信号。此试剂盒适用于大多数物种。根据不同实验中细胞类型和培养时间，0.5-2×10⁵个细胞/孔（或100微克/孔的总裂解蛋白）足以满足大多数的实验需求。为了获得最好的效果，推荐的细胞数或裂解物的浓度滴定参见图1和图2。由于caspase-7与caspase-3有相同的底物序列，该产品还能够检测caspase-7的活性。Caspase-3 Activity Assay Kit是一种荧光法检测细胞裂解液中caspase-3活性的产品。它包含一个caspase-3的荧光底物（N-Acetyl-Asp-Glu-Val-Asp-7-氨基-4-methylcoumarin或Ac-DEVD-AMC）。在检测过程中，活化的caspase-3在DEVD和AMC之间剪切该底物，使用激发波长为380 nm，发射波长为420-460 nm的荧光读取器，可以检测到产生的高荧光AMC。底物的剪切仅发生在凋亡细胞的溶胞产物中，因此，AMC产生的量是与样品中的凋亡细胞数成比例的。

Caspase-3 (CPP-32, Apoptain, Yama, SCA-1)是一个凋亡的关键执行者，因为它对许多关键的蛋白起着部分或全部的裂解作用，比如核酶多聚(ADP-ribose)聚合酶(PARP)（1）。caspase-3的活化需要经过其未激活的的酶原转变成活化的 p17 和 p12 片段的蛋白水解过程。caspase-3的剪切需要天门冬氨酸位于p1位点（2）。

Caspase-7 (CMH-1, Mch3, ICE-LAP3)被鉴定为凋亡的主要执行者（3-6）。 Caspase-7和caspase-3一样，是半胱天冬酶效应子，负责裂解下游底物，如PARP（3,5）。在细胞凋亡中，caspase-7被上游的在ASP23、Asp198和Asp206位点发生蛋白水解过程的caspases 激活，从而产生成熟的亚基（3,5）。类似caspase-2和caspase-3，caspase-7优先切割识别的序列DEVD后的底物（7）。

1. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
2. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
3. Fernandes-Alnemri, T. et al. (1995) Cancer Res 55, 6045-52.
4. Duan, H. et al. (1996) J Biol Chem 271, 1621-5.
5. Lippke, J.A. et al. (1996) J Biol Chem 271, 1825-8.
6. Cohen, G.M. (1997) Biochem J 326 ( Pt 1), 1-16.
7. Thornberry, N.A. et al. (1997) J Biol Chem 272, 17907-11.

原厂资料：

Specificity / Sensitivity

Caspase-3 Activity Assay Kit detects fluorescent AMC dye produced from cleavage of Ac-DEVD-AMC by activated caspase-3 in apoptotic cells. This kit is expected to work in most species. Depending on the cell type and the incubation time applied in the assay, 0.5 - 2x10⁵ cells/well (or 100 μg/well of total lysate protein) is sufficient for most experimental setups. For best results, cell number or lysate concentration titrations are recommended (see Figures 1 and 2). Because caspase-7 shares the same susbtrate sequence as caspase-3, this kit also detects caspase-7 activity.

Description

The Caspase-3 Activity Assay Kit is a fluorescent assay that detects the activity of caspase-3 in cell lysates. It contains a fluorogenic substrate (N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin or Ac-DEVD-AMC) for caspase-3. During the assay, activated caspase-3 cleaves this substrate between DEVD and AMC, generating highly fluorescent AMC that can be detected using a fluorescence reader with excitation at 380 nm and emission between 420 - 460 nm. Cleavage of the substrate only occurs in lysates of apoptotic cells; therefore, the amount of AMC produced is proportional to the number of apoptotic cells in the sample.

Background

Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).

Caspase-7 (CMH-1, Mch3, ICE-LAP3) has been identified as a major contributor to the execution of apoptosis (3-6). Caspase-7, like caspase-3, is an effector caspase that is responsible for cleaving downstream substrates, such as PARP (3,5). During apoptosis, caspase-7 is activated by upstream caspases through proteolytic processsing at Asp23, Asp198, and Asp206, thereby producing the mature subunits (3,5). Similar to caspases-2 and -3, caspase-7 preferentially cleaves substrates following the recognition sequence DEVD (7).

1. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
2. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
3. Fernandes-Alnemri, T. et al. (1995) Cancer Res 55, 6045-52.
4. Duan, H. et al. (1996) J Biol Chem 271, 1621-5.
5. Lippke, J.A. et al. (1996) J Biol Chem 271, 1825-8.
6. Cohen, G.M. (1997) Biochem J 326 ( Pt 1), 1-16.
7. Thornberry, N.A. et al. (1997) J Biol Chem 272, 17907-11.

注意事项：

Note: This kit contains mixed storage components. Please store this entire kit at -20°C for long term storage. Upon
first use, please allow components to thaw and then store each component as indicated on individual component labels.