Western blot: uneven transfer

1. Ranked causes

   1. Air bubbles or incomplete sandwich contact during transferCircular voids on Ponceau align with sandwich centre or roller gaps.
   2. Transfer buffer pH / methanol composition off for the membrane typeGradient or edge-weak transfer after a fresh buffer prep; pH drifts from 8.3.
   3. Transfer time or voltage too short for large proteins (or too aggressive for small)High-MW targets retained in gel; low-MW targets blow through to filter paper.
   4. Power supply current drift or loose electrode contactCurrent log unstable; other tanks with same buffer transfer evenly.

2. Recovery steps

   1. Re-assemble the sandwich under transfer buffer; roll out bubbles firmlyBubbles are the most common local void pattern.
   2. Make fresh transfer buffer; verify pH 8.3 and methanol % for PVDF vs nitrocelluloseWrong buffer composition causes edge/centre gradients.
   3. Extend wet transfer (e.g. 30 V / 90 min → 100 min) for targets >100 kDaLarge proteins often need more time; confirm with Ponceau + residual gel stain.

3. Controls first

   • Does Ponceau on the positive-control / ladder region also look patchy or voided?If yes: Transfer assembly or buffer/power — not antibody detection.

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