Factors to consider when using caspase activity assays

Caspase activation is an essential step of the apoptotic cascade that leads to cell death.

​The two main pathways through which apoptotic cell death is initiated are the intrinsic and extrinsic cell death pathways, characterized by caspase 9 and caspase 8 activation, respectively.

Assays that determine caspase activity through cleavage of biochemical substrates are a quick and convenient method to assess caspase activation in cells. However, caution is recommended when interpreting the data from these experiments due to the following factors:

  1. The substrate in a given assay is not necessarily specific to a particular caspase. As cleavage specificities of caspases overlap, reliance on a single substrate/assay is not recommended for identification of a specific caspase. Best practice is to use more than one method to confirm specific caspase activation. For example, caspase 8 activity assays should be used in combination with western blot detection of uncleaved/cleaved (active) caspase 8.
  2. The expression and abundance of each caspase in a particular cell type and cell line will vary.
  3. As the activation and cleavage of caspases in the cascade will change over time, it is important to consider the point of peak expression of the particular caspase e.g., after 3 hours, after 20 hours etc. Classification of caspases based on synthetic substrate preference does not reflect the true caspase substrate preference in vivo and may provide inaccurate information for discriminating amongst caspase activities. Thus, caution is advised in applying the intrinsic tetrapeptide preferences to predict the targets of individual caspases.

The tables below show known cross-reactivities with other caspases.

Inflammatory Caspases

CaspaseCleavage MotifInhibitor MotifCross reactivity with other caspases
Caspase 1YVAD
4?, 5
Caspase 4LEVDLEHD*5
Caspase 5WEHDLEHD*1,4
Caspase 12ATAD

*Inhibits at high concentration

Apoptotic Initiator Caspases​​​​​

CaspaseCleavage MotifInhibitor MotifCross reactivity with other caspases
Caspase 2VDVAD
3, 7
Caspase 8IETDIETD, LETD3, 6, 10
Caspase 9LEHD
3, 6, 8, 10
Caspase 10AEVD
3, 7, 8?


Apoptotic Executor Caspases​​​​

CaspaseCleavage MotifInhibitor MotifCross reactivity with other caspases
Caspase 3DEVDDEVD, LEHD*, IETD, LETD2, 7
Caspase 6VEIDDEVD, LEHD*, IETD, LETD3
Caspase 7DEVDDEVD, LEHD*, IETD, LETD1, 3

​​*Inhibits at high concentration

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