which one is first phosphorilated or total protein?
 

Dear all,

I have transferred my protein in PVDF membrane and I want to determine the expression of phosphorilated EGFR and total EGFR. Which one should I blot first? previously I have blot actin, and then phosphorilated EGFR and then EGFR. I have good result in actin but nothing in both p-EGFR and EGFR. Do the order of blotting influence the result?

Thank you for any advice

SiJohn