Caspase-3 Activity Assay Kit
货号: 5723S 品牌: CST
规格: 1Kit

价格: ¥ 7168.00

目录价: ¥7168.00

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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.
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.
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).


Figure 1. NIH/3T3 cells were treated with Staurosporine #9953 (5 μM, 5 hr) and then lysed in PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 (supplied with kit). Various amounts of cell lysate were added to assay plates containing the substrate solution, and plates were incubated at 37ºC in the dark. Relative fluorescent units (RFUs) were acquired at 1 and 4 hr.

图1. Staurosporine #9953 (5 μM, 5 hr)处理NIH/3T3细胞,然后溶解在PathScan® Sandwich ELISA Lysis Buffer (1X) #7018(试剂盒提供)中。将不同量的细胞裂解液加入到含有底物溶液的检测板中,并在黑暗、37℃下孵育。相对荧光单位(RFU)在第1和第4小时获得。

Figure 3. HeLa cells were seeded at 1x105 cells/well in a 96-well plate and incubated overnight. Cells were treated with various concentrations of Staurosporine #9953 (5 hr) and then lysed in 30 μL of PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 (supplied with kit). Cell lysate was mixed with substrate solution and incubated at 37ºC in the dark for 2 hr and relative fluorescent units (RFUs) were acquired.

图3. HeLa细胞在 96孔板中培养过夜,达到1x105 细胞/孔,经不同浓度Staurosporine #9953 ( 5 hr)处理后,溶解在30 μl 的PathScan® Sandwich ELISA Lysis Buffer (1X) #7018中(试剂盒提供)。将细胞裂解液混合底物溶液在黑暗中37℃下孵育2小时。测得相对荧光单位(RFU)。

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