Western blot: multiple bands

1. Ranked causes

   1. Primary antibody non-specific binding (concentration or clone)Extra bands fade when primary is diluted; persist on unrelated lysates.
   2. Degradation / proteolysis during lysis (missing inhibitors, warm samples)Ladder of lower-MW bands; fresh inhibitors + cold lysis cleans the pattern.
   3. Incomplete reduction leaving multimers plus monomerHigh-MW extras collapse after fresh reductant + heat.
   4. Secondary antibody binding endogenous Ig or Fc receptorsNo-primary control still shows some bands; species-switch secondary clears them.

2. Recovery steps

   1. Titrate primary 2-fold down and compare specific vs extra bandsNon-specific bands usually drop faster than the true target.
   2. Lyse on ice with fresh protease (and phosphatase) inhibitorsStops degradation ladders that look like multiple specific bands.
   3. Include a no-primary control strip to check secondary backgroundIdentifies Ig/Fc artefacts before changing primary clones.

3. Controls first

   • Do no-primary control lanes also show the extra bands?If yes: Secondary / blocking issue — change secondary species or block harder before blaming primary.

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