Hi, all - I use an older BioRad system for western blotting. The membranes I use are just a standard BioRad PVDF on a roll that I cut to size. The membranes work great all throughout the blotting process . . . unless I have to reprobe one . . .
After exposure I wash the membrane a couple time with TBS-T and then let dry out. Once dry, I re-activate with some methanol for a minute or so. Then I wash a couple times with DD H2O and put it in the 1X reprobe solution (5X solution from G Biosciences, diluted to 1X with DD H2O). The problem is, the membranes always float on top of the reprobe solution! No matter how much I poke them down or swish things around, they rise to the top again!
I know PVDF is hydrophobic, but I never have any issues with any of the other reagents (TBS-T, antibodies, milk, etc) which are also made in DD H2O - PLUS I asked a colleague who reprobes all the time about this and she said she's never had issues with membranes refusing to submerge into reprobe solution (and she uses the same exact brand I do and dilutes it in the same exact DD H2O that I do)!
So what is up!? Any suggestions??