I have transferred gel in PVDF membrane and stained with ponceau but I couldn't see any bands on the membrane, instead, the whole PVDF membrane is turned into pink color and the color is not going after prolonged wash also. I have used 30 microgram of protein and I transferred in semidry transfer apparatus. I have used the nitrocellulose membrane before that for the same concentration of protein and I could see the bands after stained with ponceau. I got this problem only with PVDF membrane. I have also activated the PVDF with methanol after transfer and stained with ponceau even if I couldn't find any bands. What might be the reason and how to see the bands and how to remove the background pink color?
activate PVDF first before going for transfer. Incubate PVDF membrane in methanol such that whole membrane is submerged for 2-3 min. Then keep membrane in transfer buffer for 2-3 min such that whole membrane is submerged. Not your transer buffer should have 20% methanol plus 0.1% SDS. keep transfer time for 60-80min with constant current of 75mA
activate PVDF first before going for transfer. Incubate PVDF membrane in methanol such that whole membrane is submerged for 2-3 min. Then keep membrane in transfer buffer for 2-3 min such that whole membrane is submerged. Not your transer buffer should have 20% methanol plus 0.1% SDS. keep transfer time for 60-80min with constant current of 75mA
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