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Smeared transfer on nitrocellulose membrane - (Jan/24/2012 )


I did two transfers in the same tank, while one membrane had perfect transfer, in the other the protein ladder was smeared across the membrane. Samples loaded on both the gels are the same (10% SDS-PAGE gels that I made). Before both the transfers I equilibrated the gels and membranes for around 5-10 minutes in transfer buffer. Transfer was done at 100 V for 90 minutes and left at 4C overnight. I prepared fresh transfer buffer with 20% methanol.
This has happened to me twice that one transfer is perfect while the second transfer is blotched . does anyone have a clue why this is happening?


It sounds like there was some sort of gap between the gel and the membrane, you could try packing the transfer cassettes tighter and rolling the "sandwich" carefully during assembly.


Thanks! Yeah I did make sure about that. Hope this doesn't happen again