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zVAD-fmk, caspase inhibitor does not inhibit. - (May/19/2007 )

Dear all,
I wanted to first confirm that caspase inhibitor zVAD inhibits caspases and my readout was by checking PARP1 cleavage (WB). I proceed as follows:
I add or (do not add) zVAD(20uM) 1 hour before I add a known apoptotic stimuli ((Etopside) 30uM) to the Raw cells. I then let cells go 1h or 3h or 6h. But it seems that addition of zVAD does not change anything.
Why would this be? Is it concentration of zVAD or Etoposide? But I checked the literature and people could even inhibit PARP1 cleavage by using less zVAD and more Etoposide.
I would be appreciated for any suggestion.



just a little question: is your VAD really z-VAD, or even VAD(Ome)?
these chemical modifications enhance the permeability of the caspase inhibitor for an efficient cellular uptake...
that is right that apoptosis induced by Eto is wel inhibited by less z-VAD-fmk
in contrast, apoptosis induced by some ligand on lymphoma cells is inhibited by only 25%...



z VAD FMK is a broad range caspase inhibitor. You would like to check for reduction in caspase -3 activity compared to your etoposide treated and untreated cells rather than PARP cleavage which is down stream of caspase -3 activation.

Also to see if z VAD FMK is working alright try to induce cell death using either cycloheximide or TNF alpha and inhibit it with z VAD FMK.

Good luck,