Best Practices for Caspase Activity Assays

The activation of caspase, an enzyme in the protease family, is a key step in the apoptotic cascade, i.e. the cell death pathway.

There are two key pathways which begin apoptosis – the extrinsic pathway and the intrinsic pathway. Both pathways are activated by a caspase, with the extrinsic pathway activated by caspase 8 and the intrinsic pathway by caspase 9.

It is possible to determine the level of caspase activity, and how much caspase activation is occurring in a sample, using an assay that measures the cleavage rate of a caspase substrate. However, when these assays are conducted the following points must be considered:

  • Each different cell line and type shows differences in caspase concentration and expression
  • As the cleavage behavior of different caspases show overlap, substrates used in caspase assays are not always specific to one certain type of caspase. For this reason, using only one substrate to analyze a specific caspase is not accurate. Instead more than one assay, using different substrates, is best. E.g. an assay to test for caspase 9 should be run alongside a western blot analysis for active (cleaved) and uncleaved caspase 9.
  • Over the course of the apoptotic cascade the activation and cleavage of caspases changes, meaning the time of peak caspase expression for a particular caspase must be taken into consideration. Classifying caspases on the basis of their preference towards the synthetic substrates used in assays is not a true reflection of a caspases behavior in vivo and can give false information about a caspase’s activity. For this reason, care must be taken when using intrinsic tetrapeptide preferences to determine the target substrates for individual caspases.

The tables below demonstrate the known cross-reactivities of caspases.

Inflammatory Caspases

Caspase Cleavage Motif Inhibitor Motif Cross reactivity with other caspases
Caspase 1 YVAD 4?, 5
Caspase 4 LEVD LEHD* 5
Caspase 5 WEHD LEHD* 1,4
Caspase 12 ATAD

Apoptotic Executor Caspases​​​​

Caspase Cleavage Motif Inhibitor Motif Cross reactivity with other caspases
Caspase 2 VDVAD 3, 7
Caspase 8 IETD IETD, LETD 3, 6, 10
Caspase 9 LEHD 3, 6, 8, 10
Caspase 10 AEVD 3, 7, 8?

Apoptotic Initiator Caspases​​​​​

Caspase Cleavage Motif Inhibitor Motif Cross reactivity with other caspases
Caspase 3 DEVD DEVD, LEHD*, IETD, LETD 2, 7
Caspase 6 VEID DEVD, LEHD*, IETD, LETD 3
Caspase 7 DEVD DEVD, LEHD*, IETD, LETD 1, 3

*Inhibits at high concentration

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: May 15, 2019 at 8:42 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Abcam. (2019, May 15). Best Practices for Caspase Activity Assays. News-Medical. Retrieved on November 15, 2019 from https://www.news-medical.net/whitepaper/20190515/Best-Practices-for-Caspase-Activity-Assays.aspx.

  • MLA

    Abcam. "Best Practices for Caspase Activity Assays". News-Medical. 15 November 2019. <https://www.news-medical.net/whitepaper/20190515/Best-Practices-for-Caspase-Activity-Assays.aspx>.

  • Chicago

    Abcam. "Best Practices for Caspase Activity Assays". News-Medical. https://www.news-medical.net/whitepaper/20190515/Best-Practices-for-Caspase-Activity-Assays.aspx. (accessed November 15, 2019).

  • Harvard

    Abcam. 2019. Best Practices for Caspase Activity Assays. News-Medical, viewed 15 November 2019, https://www.news-medical.net/whitepaper/20190515/Best-Practices-for-Caspase-Activity-Assays.aspx.

Other White Papers by this Supplier