Larger bands are mostly caused by post-translational protein modifications such as glycosylation and phosphorylation, incomplete protein depolymerization or residual multimers. Insufficient β-ME in loading buffer also prevents disulfide bond cleavage. Smaller bands usually result from protein degradation: repeated freeze-thaw cycles or lack of protease inhibitors lead to truncated proteins; alternatively, splice variants or truncation mutants may be present. Mismatched gel concentration, improper electrophoresis voltage and duration, degraded markers, and non-specific binding of primary antibodies can all cause inaccurate molecular weight reading. Adding protease inhibitors, thorough denaturation and adjusting separating gel concentration are recommended for verification.