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selection of caspase activity assay

Caspase ELISA flowcytometry protein

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#1 Xu Zhu

Xu Zhu

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Posted 28 October 2012 - 07:55 AM

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!!!

#2 gmcstay

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Posted 29 October 2012 - 06:06 AM

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





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