So I have been having a lot of issues detecting a particular protein via western blot. When I image the blot I get a lot of clear bands going all the way up the lane, almost looking like a Coomassie stain. However I have no signal at the molecular weight of my protein. Not even close. I have adjusted so many steps from my lysis buffer to type of transfer (dry vs wet). I keep getting these same non specific bands and next to no signal at my target MW, which is around 19kD. Any suggestions would be appreciate. Ive tried 15ug of protein all the way to 30 ug of protein per well. Also other antibodies work fine. GAPDH is perfectly normal.
The most likely explanations are (1) the protein of interest is not present in the extract, or has become proteolytically degraded into smaller pieces, and (2) the antibody lacks the intended specificity.
maybe you should change the purification protocol and adapt it to the protein. there are kits with various adjuvants for testing. do you know if your protein binds a linear or a conformational epitope? maybe there is a recognition problem if it is the latter…
Hi!
For solving your problem you should to give a detailed protocol or describe a condition of your probe and gel preparation, WB and antibodies.
Recently I have the same problem when testing new modified protocol for turbo-electrophoresis. Control membrane was obtained by classical protocol, but the pilot one was obtained by using PAA-gels with higher glycine concentration. The running buffer also was made with higher Gly concentration, and thus time of electrophoresis was ~30min at 300 V. Both membranes were stained with primary Ab to cleaved-caspase3 (35 and 17/19 kDa subunits). On control membrane I saw the both subunit, 37 and 19 kDa, but only big subunit (35 kDa) on the second (=pilot) membrane. I also tested Ab to yH2Ax (~12-15 kDa). I didn't saw any band below 25 kDa.
Actually, I don't know if my experience will be useful for solving your problem, but hope it will. I suppose, small proteins or their epitops may degrade during phoresis and transfer stronger then the medium/large proteins, especially by using non-classical conditions.
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