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 7 DEVD DEVD, LEHD*, IETD, LETD 1, 3

*Inhibits at high concentration

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


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