I have very difficult problem to which I need your help! I have whole cell lysates from different human lymphocyte cell suspension which are diluted in 2,5 X Laemmli buffer (Laemmli includes NaF & Na3VO4). After collecting my samples I have stored them in -20. Before loading the samples into the SDS-PAGE gel I have sonicated them to break the membrane (3 x 10 seconds with 7,5 amplitude) and then measured the protein concentration by RC DC kit (BioRad). Then I have loaded equal amount of protein in the well, so that in every well the loaded volume is a bit different depending on the concentration. Result is unequal loading, I am using b-tubulin as loading control. I have tried to correct the loading by calculating the b-tubulin signals and then calculated how much more or less I need to load my samples. But even after these corrections I end up with very unequal b-tubulin response. I have tried this many times but every time the result is the same. I have tried to load with different kinds of pipet tips and pipets but nothing helps.
Does anyone have similar problems and how have you solved them?