Hi everyone - I'm having a problem with background signal in my total protein stain using PVDF membranes and was wondering if anyone had suggestions for what is causing it and how to remedy it.

I have started to use PVDF membranes for my Western blots because they work better (the gel doesn't stick to the membrane like it does to nitrocellulose) with the gels we buy. However, the main downside is that the total protein stain (I use LICOR's Revert 520) consistently has little specks all over the protein signal. This has never been a problem with the nitrocellulose membrane total protein stain. The only difference between the two protocols is the methanol activation - I soak the PVDF membrane in methanol for 20 min before transferring. I then rinse it in transfer buffer before putting it in the sandwich. Then I soak it in methanol for 5 minutes while rocking to reactivate it after the drying step of the Revert 520 protocol.

I have attached images of what my Revert 520 total protein stain looks like on PVDF membrane vs nitrocellulose. Any suggestions would be greatly appreciated.