Problems with caspase 3 activity assay - (Feb/21/2006 )

I am trying to perform a caspase 3 activity assay in our lab.
We are using human recombinant caspase 3 (Alexis) with a fluorescent substrate to generate a standard curve.
The assay has worked fine last year in april and gave a nice standard curve for various concentrations of caspase 3.
Now I can measure activity only in one of the 8 concentrations I made.

Any idea what I am doing wrong?

Thank you very much for your help.

tamerza

In my experience, the substrates lose their effectiveness quite quickly, even when stored at -70. You can try another assay: Promega sells a number of caspase-3 kits (fluorimetric & colorimetric) or you can try analysis of caspase-3 by flow cytometry.
Good luck!

Thanks for your answers!

I am trying to give some more information:

The Enzyme was dissolved in PBS/15% Glycerol to give a final concentration of 1U/µl.

Further dilutions were made in assay buffer (10mM HEPES/KOH pH 7,4/0.1% CHAPS/5mM DTT/2mM EDTA/2mM Pefabloc/0.1% Triton X-100) to give concentrations between 0.05U/100µl and 0.6U/100µl.
100µl of each dilution were added to a 96-well plate.

10µl substrate Ac-DEVD-AMC (0.2mM in assay buffer) were added to each well.

Fluorescence was measured for 30min every 2min at exic. 360nm and em. 460nm.

To check out the substrate I tried old cell lysates that had already been tested positive and they still work fine.

I made up fresh assay buffer - no improvement.


The enzyme was fesh and as I said before, I get good caspase acitvity in the 0.2U/100µl dilution. But nothing above or below.

I am really lost!

Greets,

tamerza

I am also doing a caspase 3 assayy using a fluoregenic Ac-DEVD-MAC substrate. Could you please let me know how i can get a standard curve. Many protocls I have seen requires me to only make one pure caspase 3 dilution and taking furoscence measurements at different intervals. Is this the right method or is there any other way?

Thanks,
Suma