Why am I seeing multiple or unexpected bands on a Western Blot with the affinity purified antibody?

  • Many proteins don’t appear at their predicted molecular weights on SDS Page gels due to issues such as splicing variants, post-translational modifications (PTM), protein degredation and electrophoretic migration behavior. In particular, there are more than 300 types of PTMs that can affect the molecular weight seen on the gel.
  • It is common to run assays with dilutions of antibody that are too high and thereby lead to a lot of background noise. If you are seeing a lot of “cross-reactivity” with the purified antibody, try working with significantly lower dilutions of the antibody. Overall, most of the purified antibodies are extremely sensitive and so the minimum amount possible should be used in assays.
  • Antibodies against the peptide may recognize homologous epitopes from another protein in the sample.
  • The antibody may recognize splicing variants of the target protein.
  • The antibody may recognize cleaved fragments of the target protein at lower molecular weights.
  • The antibody may recognize aggregated dimers/trimers of the target protein at higher molecular weights.
  •  The target protein may be at a different molecular weight than previously predicted (perhaps because of reasons listed above).

Why am I not seeing any bands on a Western Blot when assaying with the affinity purified antibody?

  • The peptide sequence may correspond to an unexposed region of the native protein.
  • The protein’s conformation in the peptide region may differ from the conformation of the synthetic peptide.
  •  The target protein may not be present in the sample, or the epitopes may have been modified.
  •  The protein may not have been properly transferred to the membrane.
  • Primary or secondary antibody concentrations may require adjustment.

Next: Affinity Purification