I have been trying to transfer minigels after running SDS-PAGE this week and I keep having the problem of not seeing my MW bands on the PVDF. I am using two kinds of MW markers, one is from bio-rad, and another is the ultra low molecular weight range marker from sigma aldrich. For both markers, I cannot see the bands on the membrane, and they do not appear on the whatman paper behind my membrane either. I am also hesitant to probe my membrane with primary antibody because it is really hard to obtain, and it would be a waste of solution if there was no target on my membrane I think.
just to give you an overview of what I did; I ran samples containing a 1 kDa protein and my markers on the minigel, and then I transferred overnight (16 hours) at 30 volts using transfer buffer containing methanol. I also wet my membrane for about a minute in 100 % methanol before running, and I am pretty sure my electrodes and sandwich were set up correctly. So is there anything else that could be going wrong here ?
Lastly, I read another post on here where someone suggested using ponceau red to stain the membrane and check if the proteins were on the membrane. If I only have coomasie brilliant blue available, could I use that and successfully destain my membrane ?