#5723 Caspase-3 Activity Assay Kit
|Ac-DEVD-AMC||1 x 1 mg|
|AMC (7-amino-4-methylcoumarin)||1 x 250 µl|
|PathScan® Sandwich ELISA Lysis Buffer (1X) #7018||1 x 30 ml|
|Caspase Assay Buffer (2x)||1 x 30 ml|
|DTT (Dithiothreitol) #7016||1 x 192.8 mg|
|細胞抽出物中のCaspase-3 の活性を検出する蛍光分析キットです。Caspase-3 の検出に必要な蛍光基質 (N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin またはAc-DEVD-AMC) を含みます。活性型Caspase-3 がこの基質をDEVD とAMC の間で切断し、高い蛍光性を持つAMC が生成され、これを蛍光リーダーで読み取ることでCaspase-3 の活性を測定します。|
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.
Figure 2. NIH/3T3 cells were seeded in a 96-well plate at 1x105 cells/well or 5x104 cells/well, and then treated with Staurosporine #9953 (5 μM, 5 hr) and then lysed in 30 μl PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 (supplied with kit). Cell lysate was added to assay plates containing the substrate solution, and plates were incubated at 37ºC in the dark. Relative fluorescent units (RFUs) were acquired at 0, 1, 2, 4, and 6 hr.
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.
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).
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