I'm new to this forum and I'm using a Biorad transblot machine to transfer the proteins from my gel to the membraben.
I am using PVDF for the membrane. PVDF treatment includes 10-15 seconds soak in methanol and then soaking in transfer buffer for at least 15-20 minutes. I just leave it in there until the SDS-PAGE gel is done.
After that I do the transfer usually at 10 V for 30 minutes, but that hasn't been working so I tried 15 V for 30 minutes today.
After that I stained the gel with coomassie blue for ~2 hours and many bands showed up. The marker was gone and the marker I can see on the PVDF, but the gel is FILLED and I mean FILLED with protein bands.
How can I ensure a complete transfer and is there a way to make sure everything is transferred without having to remake PAGE gels all the time? Thanks guys, I just found this forum and it's great.