I am looking for some advice. I am trying to detect FGFR1 (~92kDa according to the antibody specification sheet) in some cell lysates. I ran a 12% gel and had excellent transfer to a PVDF membrane. The lanes had 2 of our samples as well as 3 positive controls (homogenate and membrane samples from another lab-we don't know too much about them or how they were handled) and protein standards (loaded undiluted and unheated per the instructions for the ladder). I got strong specific bands with zero background in the positive control lanes and weaker bands in my sample lanes but they were all at the wrong molecular weight (15kDa)!
Tech support for the primary antibody suggested heating samples at 60 degrees for 15 min rather than 95 degrees for 5 minutes like I did to avoid possible protein aggregates that would run faster because of a skewed mass:charge ratio.
A few people also suggested that perhaps the protein was breaking down (it was refrozen/thawed) but it seems suspicious that the protein would degrade similarly in all the samples and show up at the exact same moleuclar weight in all cases)
The primary antibody (monoclonal anti-fgfr1 IgM raised in mouse) is from Millipore, secondary is from Santa Cruz (IgM anti-mouse HRP) and I used Millipore Luminata Forte HRP substrate(very sensitive).
I wanted to see if I'm missing something before I use up more samples/antibody with another gel and western.
Thanks so much!