Hi! I have several years of experience with Western blot and have recently developed issues with variable protein loading.
Briefly, I am using lysates from cultured cells using either a RIPA buffer or a tris-based lysis buffer for phospho-proteins. I am using pre-cast gels and buffers (invitrogen). I have used both a Bradford (Bio-Rad) and Lowry (homemade buffers) for protein determination and run my standards and samples in duplicate so I can average the results. I don't think I am losing the sample during loading as I have not previous had this problem and think my technique is okay.
One big change in my method has been the preparation and reading of the protein concentration. I was preparing my Bradford samples in disposable cuvettes for a spectrophotometer that broke a while back. I adapted the protocol and now prepare the samples in microfuge tubes, place samples into wells on a standard 96 well plate and read the results on a Polar star Omega plate reader.
I can't identify any obvious problems, but my tubulin control blots are ugly and show clear differences in between lanes.