Hello everyone. I am in the middle of performing western blotting experiments measuring expression of p-S6(Ser235/236) protein in total cell lysate.
Originally, I was quantifying my data using ImageJ, and normalizing protein expression compared to the housekeeping gene on the same blot. (Ex: HSP60)
However, I was notified by my PI that she read a paper where the researchers compared expression of p-S6 to S6 protein on the same blot. It sounds like the researchers simply stripped the blot and re-probed with unphosphorylated protein since they are both the same size.
Which approach is the best way to normalize expression of phosphorylated protein on a single blot? I am considered that stripping and re-probing the same blot will cause loss of protein & unwanted retention of the phosphorylated protein.
The best option is to conduct two-color Western blots with two primary antibodies from different species and different fluorescently labeled secondary antibodies to simultaneously detect and discriminate the two signals.
As primary antibodies use a phospho-specific antibody and a pan-specific antibody that recognizes the target protein regardless of its modification state.
Phospho-specific signals are then normalized against the total level of the target protein.
See also:
https://www.researchgate.net/post/How_to_detect_phospho_and_total_form_of_a_protein_using_western_blot