Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

selection of caspase activity assay

Caspase ELISA flowcytometry protein

  • Please log in to reply
1 reply to this topic

#1 Xu Zhu

Xu Zhu


  • Members
  • Pip
  • 1 posts

Posted 28 October 2012 - 07:55 AM


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?

#2 gmcstay



  • Active Members
  • Pip
  • 5 posts

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.


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


Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.