Local blank patches / air bubble imprints (Western Blot)

Question

Accepted Answer

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: 1) Air bubbles trapped between gel and membrane during transfer 2) Filter paper or sponges were not fully soaked 3) Transfer sandwich was shifted or seated unevenly 4) PVDF membrane was not properly pre-treated (not methanol-activated) Solutions: 1) When assembling, use a roller or a pipette to gently push out all air bubbles 2) Fully pre-wet all transfer consumables (paper, sponges) in transfer buffer 3) Check membrane and gel orientation; ensure they are properly aligned 4) Activate PVDF in methanol for ≥15 seconds, then equilibrate in transfer buffer