polyclonal antibody doesn't pick up my protein during western!? - (May/23/2006 )

Hi all...I'm really really at my wits' end...

We're trying to express a ~10kDa human protein in Ecoli. SDS-PAGE shows a big fat band around the correct MW comparable to commercially purchased positive control(so far so good). However, Western Blot gave a blank...it didn't even pick up our purchased positive control! Naturally, we thought...it may be people errors or the antibody. So we tried it a couple more times, even got different people to repeat the exp, same result. so we bought 3 more different commercially available polyclonal antibodies for our protein. Turned out 2 of the 4 antibodies picked up the positive control and the other 2 didn't. One of the ones that picked up the positive control also picked up a weak band slightly smaller than the big fat band seen on the SDS-PAGE. What's happening! Help!

also, we sequenced the gene at cloning, so i don't think that's the problem...

thanks in advance!

Maybe your antibodies were raised for native protein and reduced sample has so different conformation that antibodies won't pick it up?

No, same results with reduced andnon-reduced samples =( *cry*

some antibodies require very stringent conditions (salt concentration, presence and concentration of detergent, pH, etc) to work properly. have you checked with different antibody concentrations (stronger and weaker)? did the antibody come with a fact sheet that might give you an idea as to the conditions necessary for use in various procedures?

After much frustration, someone had the good sense to dish out $500 and sent that big fat band on SDS PAGE to Mass Spec. Turned out that it's NOT our protein of interest -- it's an E coli ribosomal subunit! Has this ever happened to anyone before? =( Why does the host ribosome over-express and not my protein?!

many thanks in advance!