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Can't find PARP in WB

Can't find PARP in WB - Western Blot Forum

Can't find PARP in WB - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 08-03-2011, 02:00 PM
Pipette Filler
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Default Can't find PARP in WB



Hello

I have been trying to find cleaved PARP (85 kd) in some WB samples for almost 2 months now!
I came to the extreme to using 150 ug of protein, the antibody is 4 months-old and from SC and 1:100. I am using staurosporine as a positive standard, i have checked that treated cells are apoptotic, but still i can't detect the protein in my film!

Does anyone has any experince in PARP?

Thanks a lot!

David
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Old 08-20-2011, 07:14 PM
Pipette Filler
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Default Re: Can't find PARP in WB

How do you harvest your cells? There's a grad student in the lab I work at who's doing some work with this now.

Our current method is to spin down the removed media and add our lysed cells to that pellet. We vortex our lysate media-pellet mixture and then spin that down.

We treat our cells with cis-platin over a couple of days (some low microgram amount, i don't recall) and that gives us a good signal.

Our antibodies are from cell signalling technologies however, so that might be another problem.
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