Western blot: unexpected size

1. Ranked causes

   1. Post-translational modification or cleavage shifting apparent MWShift is consistent across lysates; matches known phospho/glyco/cleavage forms.
   2. Incomplete reduction / denaturation (sample buffer, DTT/βME, heat)Re-boiling with fresh reducing agent collapses extra high-MW bands.
   3. Antibody cross-reactivity to a related protein (lot / clone)Knockdown/knockout or peptide-block removes the unexpected band.
   4. Ladder mis-assigned or gel percentage wrong for the target rangeRe-plotting against a fresh ladder or higher-% gel places the band on-size.

2. Recovery steps

   1. Re-denature samples with fresh reducing agent; heat 95 °C / 5 minIncomplete reduction is the most actionable size artefact.
   2. Confirm ladder lot and gel %, then re-estimate MWRules out scale mis-assignment before biological interpretation.
   3. Test a second primary clone or peptide-block the antibodySeparates true isoform/PTM from off-target binding.

3. Controls first

   • Does the positive-control lysate show the same unexpected size?If yes: Sample prep, ladder/gel scale, or antibody specificity — not one bad sample.

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