Antibody works in immunofluorescence, but not in WB
Posted 12 July 2010 - 06:52 PM
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!
Posted 12 July 2010 - 07:21 PM
Posted 13 July 2010 - 01:19 AM
Posted 13 July 2010 - 01:48 AM
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.
Posted 13 July 2010 - 03:09 AM
"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)."