easy & quick apoptosis assay for siRNA treated cell line sample - (Mar/27/2008 )

hi,

I am a little pressed for time and wish to perform an apoptosis assay on my samples which are 'HEY cells treated with a gene-specific siRNA'..
thus looking for apoptotic changes when the gene is downregulated.
Now, after a lot of compare-n-contrast between assays...

I was thinking to start off by a straight western blot against PARP..
and if I see some difference from untreated cells(or GFP siRNA treated HEY cells), i shall reprobe against Caspase 3 or mayb cyclins for cell cycle information?
Is my thinking ok? or shd i consider annexin or tunel or elisa?
Plz suggest given the fact that i m very new to lab research.
and any special tips for western...like anti-PARP antibodies, extracts etc.

Any help will be highly appreciated.

Thanks,

hi,

I am a little pressed for time and wish to perform an apoptosis assay on my samples which are 'HEY cells treated with a gene-specific siRNA'..
thus looking for apoptotic changes when the gene is downregulated.
Now, after a lot of compare-n-contrast between assays...

I was thinking to start off by a straight western blot against PARP..
and if I see some difference from untreated cells(or GFP siRNA treated HEY cells), i shall reprobe against Caspase 3 or mayb cyclins for cell cycle information?
Is my thinking ok? or shd i consider annexin or tunel or elisa?
Plz suggest given the fact that i m very new to lab research.
and any special tips for western...like anti-PARP antibodies, extracts etc.

Any help will be highly appreciated.

Thanks,

If you have access to flow cytometry facilities, I would suggest the easiest way would be to divide your cells up into three and stain them for:
Annexin-V
z-vad-fmk-FITC (detects activated caspases by flow)
PI staining of fixed cells (gives you cell cycle, as well as sub-G1 apoptotic cells).

This should provide a bunch of information and the protocols are pretty easy. Plus you get all you answers that day, and the cell by cell assays by flow may pick up smaller changes in the cell population than western blotting.

If you do want to go with the western, PARP and activated caspase 3 are good ones to pick. You can run one gel, slice it up and probe for PARP (~85/110 kDa) , active casp 3 (25kDa) and a loading control eg. B-actin (42 kDa I think) - so no messy reprobing required.

Its nice to have all your targets separated by size so much - don't get used to it though! I recommend Cell Signaling antibodies for the westerns.

Thank you so much for your reply.
Any recommendations for the Annexin V/PI staining kits?
Is it better than the APO-BrDU Tunel Assay kit?

Thanks,