Caspase 3 Colorimetric Substrate (N-acetyl-Asp-Glu-Val-Asp-pNA (p-nitroanilide)) (CPP-32, Apoptain, Yama, SCA-1)

Colorimetric substrate for Caspase-3.\n\nActivation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. See Caspase-3 Apoptosis Detection Kit C2087-20 which provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on detection of cleavage of substrate DEVD-pNA (p-nitroanilide).\n\nUpon proteolytic cleavage of the substrate by caspases, free pNA is detected at a wavelength of 400nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the increase in Caspase-3 activity.\n\nSequence: \nAc-Asp-Glu-Val-Asp-pNA\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.\n\nProtocol:\n1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.\n2. Count cells and pellet 1-5x10e6 cells.\n3. Resuspend cells in 50ul of chilled Cell Lysis Buffer (C2086-10F) and incubate cells on ice for 10 minutes.\n4. Centrifuge for 1 min (10,000 x g).\n5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice.\n6. Assay protein concentration.\n7. Dilute 50-200ug protein to 50ul Cell Lysis Buffer for each assay.\n8. Add 50ul of 2X Reaction Buffer (C2086-10F1) containing 10 mM DTT to each sample.\n9. Add 5ul of the 4mM C2087-21 (200uM final conc.) and incubate at 37C for 1-2 hours.\n9. Read samples at 400 or 405nm in a microtiter plate reader or spectrophotometer using a 100ul micro quartz cuvet, or dilute sample to 1ml with Dilution Buffer using a regular cuvet.

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SPECIFICATIONS

Catalog Number

C2087-21

Size

200Tests

Form

Supplied as a liquid.

Purity

~98% (HPLC)

References

Product Specific:\nCollin C., et al. (2001) J Biol Chem 276:37602-37611.\nDatta R., et al. (2000) J Biol Chem 275:31733-31738.\nDing H.F., et al. (2000) J Biol Chem 275:38905-38911.\nDoostzadel J. et al. (2000) J Biol Chem 275:25336-25341.\nFujii T., et al. (2000) J Biol Chem 275:7574-7582.\nGhatak S. et al. (2002) J Biol Chem 277:38013-38020.\nGomez-Angelats M., et al. (2000) J Biol Chem 275:19609-19619.\nKown, M.H., et al. (2002) Circulation 102:228-232.\nShain, K.H., et al. J Immunol 168:2544-2553.\n\nGeneral:\nXiang, J., et al., Proc. Natl. Acad. Sci. USA, 93:14559 (1996) \nNicholson, D.W., et al., Nature, 376:37-40 (1995) \nLazebnic, Y.A., et al. Nature, 371:346-349 (1994)