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Antibody works in immunofluorescence, but not in WB - (Jul/12/2010 )

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!


Some antibodies only work in specific assays (such as immunofluorescence) and there is nothing you can do to make it work in others (such as western blotting). I don't supposed this is a phospho-specific antibody?? If so, the one big thing is to not use milk but rather BSA for blocking and blotting. If not, the epitope this antibody most likely recognizes requires the structure of the protein which is maintained by fixation but is destroyed by denaturing conditions for western blotting. Your best option, to see if this is the case, is run a non-denaturing gel but there is nothing you can do to make it work for western blot. Sorry...I know it's not the answer you were hoping for.


With Immunostaining, you can't tell if a protein is in monomer/dimers/trimers etc. but with Western you could. Did you use the correct percentage of gel that captures all proteins across a wide molecular weight range? I am just wondering when you said it never worked, whether you mean you see no bands at all or did you see some bands but not at the size you expected them to be...


I second what rkay says.

Is your antibody specified to work on WB? If not, it probably just wont work. As rkay said the epitope the Ab recognises might be destroyed by denaturing SDS-PAGE.

Do you have a positive control for your WB? Do you have the recombinant protein? A "quick" way to test antibodies for WB is by ELISA. Coat the plate with normal protein, and boiled protein (should destroy it as for WB, even maybe boil it in loading buffer). Then incubate with your antibodies. The normal protein should be positive, if the boiled protein isn't recognised, you antibody will not work for WB.

Hope this helps.

-almost a doctor-

@rkay: its not a phospho-specific antibody. Its against cadherin. So that also answers WYF's question that its not a problem with gel percentage. The most weird thing is that the people who developed this antibody made it to work against western blot, in a paper published in 1998. I emailed those authors and they forgot everything about that antibody. This is the all the description from that paper. Do you see anything interesting?
"For immunoblotting, embryos at stage 14 were lysed with Laemmli’s sample buffer and boiled for 5 minutes. The samples were
run in a 7.5% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The blots incubated with appropriate
antibodies were processed for chemiluminescence with the ECL detection system (Amersham)."