Certain regions of the membrane show no signal at all, forming round or irregular blank zones; often due to transfer failure in those areas while surrounding bands look normal.
Common Causes
1Air bubbles trapped between gel and membrane during transfer
2Filter paper or sponges were not fully soaked
3Transfer sandwich was shifted or seated unevenly
4PVDF membrane was not properly pre-treated (not methanol-activated)
Solutions
1When assembling, use a roller or a pipette to gently push out all air bubbles
2Fully pre-wet all transfer consumables (paper, sponges) in transfer buffer
3Check membrane and gel orientation; ensure they are properly aligned
4Activate PVDF in methanol for ≥15 seconds, then equilibrate in transfer buffer