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The volumes in the protocol for ab102497 looks to be for a 96-well plate. Are there any reported problems with decreasing volumes for use in a 384-well plate (if this has been explored)? Also from the website it is not clear what the concentration of Z-VAD-fmk is used. What is the stock concentration of Z-VAD-fmk? Just to clarify the 1uL Z-VAD-fmk into 49uL H20 is used for a completely inhibited control?
Asked on Mar 15 2012
Thank you for your inquiry.
1) We have not looked at decreasing volumes for 384-well plates. You may be
able to use these, but it will depend on the amount of caspase in the samples
and hence signal strength can be impacted.
2) The Caspase Inhibitor, Z-VAD-FMK is 2 mM. I have since updated the online datasheet.
Hence 1 ul in 50 ul would result in 40 uM; which should be adequate for
recombinant Caspases. If you're looking at cell lystaes, you should probably
increase Z-VAD-FMK final to about 2-5x.
The updated component list will look like this, but it takes a few days to update:
Components 1 kit
2X Reaction Buffer 1 x 10ml
Active Caspase-9 1 x 100 units
Caspase Inhibitor, Z-VAD-FMK (2 mM) 1 x 10µl
Caspase Substrate LEHD-AFC (1 mM) 1 x 0.5ml
DTT (1 M) 1 x 100µl
I hope this information helps. Please contact us with any other questions.
Answered on Mar 15 2012