Antibody works in Immunofluorescence but not in Western blot

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jangajarn
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Antibody works in Immunofluorescence but not in Western blot

Post by jangajarn » Tue Jul 13, 2010 2:54 am

Hello everybody,
I have a problem I've been banging my head for the past 5 months. This particular monoclonal antibody works perfectly fine in detecting its antigen in immunofluorescence. But when I try to detect the protein by western blotting, its never worked. I tried different things: lysis buffers like SDS (laemelli), RIPA, non-ionic detergents, boiling & moderate heat (37C for 30 mins), new stock of antibodies, TBS & PBS buffers. I use denaturing conditions and transfer proteins to PVDF; use milk as buffer; and use Supersignal West Pico (Thermo) as a susbstrate for chemiluminescence. As last couple of options, I am trying to do run a denaturing blot and use a highly sensitive substrate. Did anybody have such problems? What could be happening?
Thanks for your time and inputs!

roxanalibe
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Post by roxanalibe » Tue Jul 13, 2010 4:54 pm

sometimes the antibodies made against the native proteins, and easy to use for IF or FACS could be bad to recognize the same protein in denaturating condition (WB); moreover there are antibodies that work very well in FACS but bind non-specific to many denaturated proteins, so they cannot be used in WB.
Did you try to put on the blot the positive control that they recommend in the data sheet of the antibody?

Good luck,
R

roxanalibe
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Post by roxanalibe » Thu Jul 15, 2010 10:12 am

if it helps, i asked around and apparently we really have in the lab antiboodies that recognize the 3D structure of the protein, so the epitope is not linear, it is impossible to use them for denaturated protein.

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