1 reply to this topic

[Xu Zhu]

Hi,

I need to quantify the caspase (3, 8 and 9 respectively) activity in cell lines treated with different concentration of drugs for different time points. So I need a quantify or semi-quantify assay with good accuracy/linearity and relatively high-throughput.
I have looked through relevant kits, including flowcytometry based assay, ELISA (colorimetric and fluoroscent), and substrate reaction based assay (e.g.  based on labeled substrate Ac- DEVD-pNA or Ac-DEVD-AMC).

I am really struggling about which assay to use. If any of you had experience with any of the assays, could you please provide some clues?
THANK YOU!!!

[gmcstay]

Hello Xu Zhu,

quantifying activity of specific caspases is not as simple as it first seems.  The available substrates are not specific for each caspase.  The "specific" substrates for caspase-8 and caspase-9 are also cleaved very well by caspase-3.  In most cases you are simply measuring caspase-3 activity with these assays.  

There are some activity based probes available.  One is biotinylated-VAD-FMK, which is commercially available, and there are some derivatives made by the laboratory of Matthew Bogyo.  You can do a streptavidin pull-down and determine which caspase has been bound (indicating activation) by western blot.

I had some success with immunoprecipitating caspases and determining activity associated with the immunoprecipitated caspase.  I only did this in cytosolic extracts activated with cytochrome c and dATP to engage the apoptosome pathway.  I never tried it in cells I treated with an apoptotic stimuli.  

Here are the papers my comments are based on.

http://www.nature.co...l/4402260a.html
http://www.nature.co...ll/ncb1340.html
http://www.sciencedi...074552112000208
http://www.ncbi.nlm....les/PMC3365438/

Good luck and let me know if you have any more questions.

Gavin