Recently, I'm having difficulties with my Western-blots and lectin-blots. Everything looks fine, until I reveal the membrane and it looks like it is divided in two halves, with the upper half appearing a lot darker than the lower half. This has been happening for a month, and previously it didn't happen.
I use 10% SDS-PAGE gels to do the Western/lectin blot. I use PVDF membranes, I activate them with methanol and then put them in transfer buffer prior to assembling the sandwich. I transfer the gel overnight at 30V 4ºC, and block the membrane with 3% BSA in TBST for an hour. For the blocking, I used a 50 mL falcon and a roller, and also small boxes increasing the volume to make sure it is completely covered, but it keeps happening this issue.
Also, using the transfer tank and the PVDF membrane from another group did not resolve the problem.
Could someone help me to solve this problem? Thank you!
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