Caspase 3 Activity Assay 细胞凋亡相关

• Specifically detect caspase 3 activity: Detect natural and recombinant human caspase 3 activity - not other caspases - using the caspase 3-specific anti-CPP32 monoclonal capture antibody
• Achieve highly sensitive detection of caspase 3 activity: Detect extremely low levels of caspase 3 activity in lysates using the kit’s fluorescent substrate in an immunosorbent enzyme assay
• Screen multiple samples simultaneously: Take advantage of the kit’s convenient 96-well microplate format when screening multiple samples
• Store lysates for extended periods: Since the same buffer serves as both lysis and reaction buffers, you can immediately assay lysates or store them for subsequent analysis

The initiation of all intracellular events seems to be characterized by the activation of specific proteases called caspases (cysteinyl-aspartic-acid-proteases). Biological function of Caspase 3 (CPP32, Yama, Apopain) activation seems to play a key role during the apoptotic process. Activated CPP32 is responsible for the cleavage, and therefore the breakdown, of several cellular components related to DNA repair and regulation. Cleavage sites of different caspases, including CPP32, are described.

1. Coating Buffer, 10x, 5 ml
2. Anti-Caspase-3, 20x, 500 µl monoclonal antibody from mouse
3. Blocking Buffer, ready-to-use, 25 ml
4. Incubation Buffer, 5x, 50 ml
5. DTT, 100x, 400 µl (1 M) stock solution
6. Substrate Solution Ac- DEVD-AFC, 20x, 650 µl (1 mM) stock solution in DMSO
7. AFC, 150 µl (1 mM) Stock Solution in DMSO
8. Positive Control, 250 µl lysate from apoptotic U937 cells
9. Microplate, 12 modules of 8 wells each
10. Adhesive Plate Cover Foils, 2 sheets

The Caspase 3 Activity Assay is a fluorometric, immunosorbent enzyme assay (FIENA) for the specific, quantitative in vitro determination of caspase 3 activity in microplates. The test principle is shown in Figure 1.
Caspase 3 is captured from cellular lysates by a monoclonal antibody. Following the washing step, substrate is added that is cleaved in proportion to the amount of activated caspase 3. Due to proteolytic cleavage of the substrate, free fluorescent AFC is generated. Free AFC is fluorometrically determined at l_max = 505 nm. The developed fluorochrome is proportional to the concentration of activated caspase 3, and can be quantified by a calibration curve. The assay time is approximately 5 hours.



Figure 1: Schematic showing the principle of the Caspase 3 Activity Assay.
Ac-DEVD-AFC = Acetyl-Asp-Glu-Val-Asp-7-amido-trifluoromethyl-coumarin
AFC = 7-amido-4-trifluoromethyl-coumarin

Figure 2: Apoptosis induction in U937 cells following exposure to 4 μg/ml camptothecin for different time intervals at 37°C. Lysates were analyzed for caspase 3 activity and fluorescence was plotted versus time. Additionally, an aliquot of the same cells was analyzed for Annexin-V binding in parallel.