Additional or Wrong-Sized Bands Appearing (Western Blot Immunodetection)

Question

Accepted Answer

Unexpected bands appear at molecular weights different from target protein. Multiple bands present instead of single expected band, or band appears at incorrect size. Common causes: 1) Primary antibody lacks specificity and recognizes off-target proteins 2) Protein degradation due to insufficient protease inhibitor or sample not kept on ice 3) Post-translational modifications: glycosylation, phosphorylation, ubiquitination alter molecular weight 4) Protein cleavage produces fragments of different sizes 5) Cross-reactivity with homologous protein family members Solutions: 1) Verify antibody specificity: test in knockout/knockdown cells, confirm absent bands in negative control tissue, use alternative antibody to same target 2) Add fresh protease inhibitor cocktail and phosphatase inhibitors to lysis buffer; prepare lysates on ice immediately after harvest 3) Consult literature for known post-translational modifications of target protein; use phosphatase treatment or deglycosylation enzymes to confirm modification 4) Run positive control lysate known to contain intact protein; compare band pattern to published data 5) Use antibody validated with Western blot images showing single band at expected size; select monoclonal antibody for higher specificity