I'm trying to determine caspase-3 activity of cultured cells after treatment using the caspase-3 substrate (Ac-DMQD-AMC (Acetyl-Asp-Met-Gln-Asp- amino-4-methylcoumarin), fluorogenic), but the assay failed.
Does anyone have a protocol that works for such assay?
thanks!
Ok, I might be wrong but I think that Ac-DMQD-AMC is not cell permeable... So that is what you need to do:
Treat your cells (6-well plate or 10cm dish) at the time point of interest put the cells on ice, lyse 10 min with a conventional lysis buffer (eg. 50mM Tris ph7.5, 150mM NaCl 0.5% Triton X100) but no!! protease inhibitors. Spin down unbroken cells and debris 10min at 10.000 rpm.
Incubate the cytosol fraction with Ac-DMQD-AMC. This is best done in a black 96 well plate... incubate from 10 to 30 min at RT and measure...
I would also include a positive control, e.g. Staurosporin works always.
Timings and amounts must be tested with your sample...
Best
Patrik
These assays normally work very well, maybe a little more background information would help the troubleshooting (Detection method: Microscope, fluorescence spectrometer... Sample and sample size... ) Also Caspase 3 cleaved or PARP cleavage by western blot work very well. Do you see visual signs of apoptosis with your cells?
Thank you Patrick and Mingning.
Patrick, I'm using fluorescence to detect caspase-3/7 activity, with the substract I've mentioned above. But the samples (treated with curcumin) had the same reading as the blank.
Is the first time I'm performing this assay... And I tried permeabilizing and lysing the cells. I believe that perhaps the buffer is not adequate, but I didn't find a recipe.
Do you guys lyse the cells for this?
Ok, I might be wrong but I think that Ac-DMQD-AMC is not cell permeable... So that is what you need to do:
Treat your cells (6-well plate or 10cm dish) at the time point of interest put the cells on ice, lyse 10 min with a conventional lysis buffer (eg. 50mM Tris ph7.5, 150mM NaCl 0.5% Triton X100) but no!! protease inhibitors. Spin down unbroken cells and debris 10min at 10.000 rpm.
Incubate the cytosol fraction with Ac-DMQD-AMC. This is best done in a black 96 well plate... incubate from 10 to 30 min at RT and measure...
I would also include a positive control, e.g. Staurosporin works always.
Timings and amounts must be tested with your sample...
Best
Patrik
Patrick, thanks a lot.
I'm gonna try with the lysis buffer as you recomended.
kind regards!
Patrick, do you have a reference paper for the protocol you wrote above? I tested it and it worked (thank you so much), so I would like to cite it in my next paper.
Thanks again.
This is not an asnwer but a question
I am also having some problem with my readings using N-Ac-DEVD-N-MC-R110 caspase 3 substrate (Fluorescence assay).
I just want to find out which method is better the Fluorescence or colorimetric method using (Ac-DEVD-pNA) for determination of caspase 3 activity in cell lysates.
Thanks
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