I have read somewhere that membrane proteins do NOT like to be boiled before loading in SDS PAGE (
Lamelli sample buffer used (161-0737)).
Indeed, I am working with a membrane protein and I don't get any signal at all when I load in SDS PAGE and then I transfer proteins from the gel to PVDF membrane (semi dry transfer). However, if I blot the proteins directly into PVDF membrane I got a signal (chemiluminescent and flurorescent substrats).
Maybe the problem is the protein aggregation because they are boiled, so thay can't migrate very well in the gel or the transfer is not efficient.
But I don't boil the proteins, how can I prevent any protease activity ? By protease inhibitors adding ?
I am gonna try with different treatment like 70°C, 90°C and no heating/boiling at all.
Thanks for your help !