Need help with Caspase WB - (Mar/17/2010 )
I have transfected A549 cells with an siRNA against my protein of interest. I see that the cells are dying and have done flow cytometry to verify with annexin V and have also do TUNEL. I have looked at p53, Bax levels and they are up. But, I have been trying to see either Caspase 3 or PARP-1 have been cleaved. For both western blots, I do not see anything. Can someone help me with the western blot or suggest an alternative protein to look at??? I appreciate the help.
Help!! on Mar 18 2010, 12:17 AM said:
do you mean you do not see any cleavage or you do no see anything at all?
If you don't see any caspase3 cleavage then I doubt apoptosis is occuring!
maybe you can try posting your blots and more people can help?
I would make sure you are using an antibody that shows both full-length and cleaved forms of caspase-3 and PARP. That way you can see both loss of the full-length form and appearance of the cleaved form with your siRNA.
For PARP it is easier to see the larger cleaved form at 89kDa than the smaller 24kDa band.
I would also run a positive control on your gels, maybe your cells treated with an apoptotic inducer such as STS or etoposide to show caspase-3 and PARP cleavage?