selection of caspase activity assay - (Oct/28/2012 )
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 o
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?
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.