Bands at Lower Molecular Weight from Protein Cleavage (Western Blot (Unexpected Multiple Bands))

Question

Accepted Answer

Distinct bands appear at molecular weights corresponding to known cleavage products or activated forms of the protein, rather than the full-length form. The fragment pattern may be specific and reproducible, distinct from degradation smearing. Common causes: 1) Physiological protein cleavage produces active fragments (e.g., caspases, procollagens) 2) Proteolytic activation is part of normal protein function in the biological sample 3) Antibody epitope is located on a cleaved fragment rather than full-length protein 4) Different cell states or treatments induce specific cleavage events Solutions: 1) Check literature and protein databases to identify known cleavage sites for your target protein 2) Verify antibody datasheet to confirm which domain contains the immunogen/epitope sequence 3) Use antibodies targeting different regions (N-terminal vs C-terminal) to identify fragments 4) Consider this expected biology rather than technical failure if cleavage is physiologically relevant