Multiple Low Molecular Weight Bands or Smearing Below Expected Band (Western Blot (Unexpected Multiple Bands))

Question

Accepted Answer

Multiple bands appear at molecular weights lower than the expected target protein, often accompanied by smearing or streaking below the primary band. This pattern suggests progressive breakdown of the intact protein. Common causes: 1) Proteases in the sample have digested the target protein during lysis or storage 2) Insufficient or absent protease inhibitors during sample preparation 3) Lysate stored too long or subjected to freeze-thaw cycles 4) Samples processed at room temperature instead of on ice Solutions: 1) Add fresh protease inhibitor cocktail immediately before cell lysis (use 1× final concentration) 2) Process all samples on ice and minimize time between lysis and loading 3) Use freshly prepared lysate; avoid repeated freeze-thaw cycles (aliquot samples if needed) 4) Boil samples immediately after adding Laemmli buffer to denature proteases