Hi all,

lately I have been struggling a lot with getting a good signal for a specific antibody on my western blot. I am particularly worried cause this antibodies works well for other lab mates of mine but we couldn't narrow it down to the mistake i am possibly making. 

The protein of interest runs as two isoforms above and below the 188 kDa band in MES running buffer. I attached a picture of one of the several attempts I did and pretty much they all look the same.Note that lane 4th and 8th are EMPTY! That means I really don't know where the smear comes from. And most of my colleagues in the lab seem to think that this doesn't look as protein degradation.

I think it should't be a transfer issue cause if I use antibody for other high molecular weight proteins they work well and i get nice and sharp clean bands.

I tried to load less protein, dilute the antibody in milk versus BSA but nothing solved the problem.

I am trying now to load MORE protein to see if this changes something but after this and remaking again my transfer buffer I wouldn't know what do.

Any advice will be more than welcome!

Thanks a lot

Antonia