Revision 2

#5723Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
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UniProt ID:

#P42574

Entrez-Gene Id:

836

Product Information

Storage

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.

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.

Species Reactivity:

All Species Expected

Product 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, Apopain, 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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 2
#5723

Caspase-3 Activity Assay Kit

Caspase-3 Activity Assay Kit: Image 1 Expand Image
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.
Caspase-3 Activity Assay Kit: Image 2 Expand Image
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.
Caspase-3 Activity Assay Kit: Image 3 Expand Image
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.