Help me convince my boss... - Calling all western blot gurus! (Mar/10/2010 )

Ok, so protein is not my forte at all - we are primarily a DNA/RNA lab but my boss wanted me to help a junior grad student in my lab to do a western blot . I used to do them all the time and quite good at the technique. The result on the first try was very good...I think. But my boss does not "believe" it and I'm having some trouble interpreting it.

We used a mouse monoclonal antibody against TRAF6 because the boss prefers monoclonal and was hoping for a nice specific response. However, this antibody has only been published once before (in a sketchy journal) while the majority of people doing TRAF6 westerns use a mouse polyclonal from a different company (santa cruz). Most TRAF6 antibodies out there yield a product of about 58 kD whereas ours yields a 38 kD product. I guess this just depends on the specific epitope targeted...is that right? Still seems odd to me.

Anyway, the problem is that though the blot did yield nice clear bands right around 38 kD, it also yielded two heavier bands, one right beneath the other, giving an overall banding pattern rather similar to this though different sizes from what is shown here.

My question is what would make a monoclonal antibody give a nonspecific result like this? What could those bands possibly be and do they in any way invalidate the bands that seem to be the correct size (48kD)? I'm trying to convince my boss that this doesn't mean that the 38kD band, and thus the entire blot, is worthless. I'm planning to order the polyclonal antibody that everyone seems to like and see if I get the same result. I'm looking for downregulation of TRAF6 in stably transfected cell lines and this first blot did seem to indicate some downregulation, though if my boss doesn't believe it...it's not worth much. Sorry if this hasn't been detailed or coherent in some way, you can ask me any other question to help clarify. Can someone enlighten me here?

there are a number of possibilities, some depending on the size of the bands, some depending on what post-translational modifications traf6 may undergo.

they could be aggregates, they could be modified traf6, they could be other proteins with a similar epitope, they could be artifacts caused by old 2-mercaptoethanol in the sds-page sample buffer.

mdfenko on Mar 10 2010, 02:16 PM said:

since they match the expected, i don't see why not (you can confirm it by other methods (eg peptide sequencing, lc-ms)).

just because the antibody found more doesn't mean that it didn't find the right thing, as well.

sometimes you need to tweak the incubation conditions to force the antibody to be more specific.