Hello,
I am writing this hoping someone can help me. I am just starting out with BN-PAGE and I have a transfer problem. It seems each time I transfer (2 membranes at a time) in a BioRad wet transfer casette one membrane transfers well but the other doesn't. I have tried it three times and it always seems to be the same. The bad transfer always has some coumassie still left on the gel and the membrane looks as though it has been destained already in methanol but the picture I have shown is what it looks like straight after transfer (picture 2)
I am using PVDF and activating for 20s in Methanol then 1min in water then 5 minutes in cold transfer buffer. I transfer at 15V,
Hi Joshua,
you might find this discussion helpful:
https://www.researchgate.net/post/Successful-transfer-of-proteins-from-Blue-Native-Page-to-PVDF-Membrane
I have not experience in a wet system, only in dry one, and the advantage is that this last is faster than the wet.
At the next day of staining, replacing with a destaining solution with 40% methanol and 10%acetic acid, and wash the gel with this solution three times one 15 min each
Good look
Hi Joshua Paul Harvey , u need to make some alterations in destaining protocol ,as Elizabeth Reynaga said u can try that .
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