Multiple / non-specific bands (Western Blot)

Question

Accepted Answer

Bands appear at molecular weights that do not match the expected target; multiple bands present where only one is expected. Common causes: 1) Poor antibody specificity 2) Protein degradation during sample prep 3) Overloading: too much total protein per lane 4) Insufficient blocking allowing non-specific antibody binding 5) Insufficient washing Solutions: 1) Switch to a validated high-specificity antibody (check vendor knockout validation data) 2) Add protease inhibitor cocktail to lysis buffer; keep samples on ice; minimize freeze-thaw 3) Reduce loading; 10-20 μg per lane is often sufficient 4) Optimize blocking — try alternative blocker (BSA vs milk) or longer time 5) Increase wash count and time