I am new to western blot and currently I am working on detection of phosphoproteins. Please see the attached picture for the detection of total p38 proteins (which I use to normalize the phospho-p38 proteins).
May anyone suggest why there is uneven distribution in a single band and why there would be a difference in total p38 proteins in different treatments which theoretically should be the same?
And may I know if I can use GAPDH for normalisation of phosphoprotein instead of the corresponding total protein (since p38 cannot be probed after stripping so phospho-p38 and total p38 cannot be probed on the same membrane)?
I did the protein assay the same day as running gel. I quantified the proteins and boiled the proteins with sample buffer and then added 80 ug of proteins into each well. I had already tried to make sure that the sample buffer is well mixed without any visible precipitation, and vortex and pipette up and down before adding into the well. I ran the gel with 150V for 2 hours and transfer by semi-dry method with PVDF membranes. I had tried to eliminate the bubbles by rolling even layers with test tube.
Thank you very much for your help!