I cannot detect Parp protein after total protein isolation
Hello everyone;
I cannot detect the Parp protein after total protein isolation. I can see my cells die morphologically after a novel drug. This death was confirmed by cell death experiments. However, I cannot see parp and cleaved parp after western blot. I don't think the problem is about western blot transfer. I think the problem is about protein isolation.
Since the presence of floating cells is very high in protein isolation, I collect the medium, centrifuge and apply ripa to the pellet.
I can solve this problem and I need a total protein isolation protocol that you know works.
Thanks.
#proteinizolasyon #parp #cparp
In my experiments I also used RIPA buffer for lysing the cells (HepG2). I wanted to detect a specific protein in the lysate via western blot analysis, however, I could not detect the endogenously expressed protein because it was not produced in sufficient amounts I suppose. Maybe this could be the case with your protein too.
I could only detect the protein when I transfected the cells with a fitting plasmid, so that they would express the desired protein in excess.
Are you sure that the antibody you use has access to its binding region on the PARP? If you do a denaturing gel prior to blotting, this should not be problematic I think. Do you use secondary labelled antibodies? How do you detect your signals on the blot?
Hi Adrian Marc Hauri
My first question is, have you checked your extracted protein to check house keeping protein expression like B-actin, GAPDH ?
If you got the band of house keeping protein and only you have problem in getting PARP band than, problem may relies on your first and second antibody not in the protein extraction method.
If you are not getting any of the protein band in western blotting than you should think from beginning. You told that, you collect all the dead cell floating in the medium. That is important for the protein quantification. and do you use protease inhibitor in protein lysis buffer? How about your protein quantification data? You should think from different aspect if you are not getting result. My suggestion is do not stocked in protein isolation method only. There are several reasons behind not getting bands in western blotting.
Best wishes
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