Multiple Bands or Smear Above Expected Molecular Weight (Western Blot (Unexpected Multiple Bands))

Question

Accepted Answer

Multiple bands or a smear/streak appear at molecular weights higher than the predicted size of the target protein. This pattern is characteristic of post-translational modifications adding mass to the protein. Common causes: 1) Glycosylation adds carbohydrate groups of variable size to the protein 2) Other post-translational modifications such as phosphorylation or ubiquitination 3) Boiling denatures glycoproteins causing aggregation and altered migration 4) Heterogeneous modification patterns create multiple molecular weight species Solutions: 1) Use un-boiled lysate for glycoproteins (heat samples at 37°C for 30 min instead of boiling) 2) Consult literature to confirm expected post-translational modifications for your target 3) Treat samples with deglycosylases (e.g., PNGase F) to confirm glycosylation as the cause 4) Adjust gel percentage to better resolve modified species (use lower percentage gels for larger proteins)