Additional Bands or Wrong Molecular Weight (Western Blot Immunodetection)

Question

Accepted Answer

Blot shows unexpected extra bands at different molecular weights than predicted, or expected band appears at wrong size. Multiple bands may appear where single band is anticipated. Common causes: 1) Nonspecific primary antibody cross-reacting with off-target proteins 2) Protein degradation due to insufficient protease inhibitor concentration or sample preparation at room temperature 3) Post-translational modifications (glycosylation, phosphorylation, ubiquitination) altering protein molecular weight 4) Protein cleavage generating fragments or isoforms of different sizes Solutions: 1) Verify antibody specificity using knockout/knockdown cell lysates or tissue lacking target protein; use alternative validated antibody 2) Prepare cell lysates on ice; add fresh protease inhibitor cocktail and phosphatase inhibitors immediately before lysis 3) Consult published literature and protein databases for known post-translational modifications affecting molecular weight 4) Run positive control samples with known protein size; use deglycosylation enzymes (PNGase F) if glycosylation suspected