Multiple Bands or Non-specific Binding (Western Blot (CST Guide))

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Western blot shows multiple bands instead of a single expected band. Extra bands may appear at different molecular weights than predicted, or background signal throughout the lane is elevated. Common causes: 1) Protein degradation due to missing or insufficient protease inhibitors: leupeptin (1.0 µg/ml) and PMSF not included in lysis buffer 2) Old lysate samples: protein degradation products accumulate over time, even at -80°C storage 3) High protein loading concentration causes excess signal and non-specific bands with sensitive antibodies 4) Post-translational modifications (glycosylation, SUMOylation, ubiquitylation, phosphorylation) cause subset of protein to migrate differently 5) Tissue samples contain multiple isoforms or splice variants migrating at different molecular weights 6) Wrong primary antibody dilution buffer: BSA produces higher background compared to non-fat dry milk for some antibodies Solutions: 1) Add protease inhibitors to lysis buffer: leupeptin (1.0 µg/ml) and PMSF, or use Protease Inhibitor Cocktail (100X) #5871 or Protease/Phosphatase Inhibitor Cocktail (100X) #5872 2) Use fresh lysate samples; if long-term storage is necessary, store at -80°C and minimize freeze-thaw cycles 3) Reduce protein loading amount if working with highly sensitive antibodies to decrease non-specific bands 4) Verify isoforms and splice variants on UniProt database; check antibody Specificity/Sensitivity section for isoform reactivity 5) Use 5% non-fat dry milk in 1X TBS/0.1% Tween-20 for primary antibody incubation to minimize background (unless antibody datasheet specifies BSA) 6) Check PhosphoSitePlus for PTM sites; use PNGase F treatment to confirm if multiple bands result from glycosylation