Why Do Dual Bands Reappear After Antibody Stripping in Western Blot When Probing for Closely Migrating Targets?
Western blotting is widely employed to assess the expression of specific proteins in biological samples. When two protein bands migrate closely on the membrane, a common strategy involves detecting one target first, followed by antibody stripping and reprobing for the second target. However, the reappearance of both bands during the second detection may be attributed to the following factors:
Incomplete Antibody Stripping
The antibody stripping procedure may not have fully removed the initial primary and secondary antibodies, resulting in residual signal from the previously detected target upon subsequent exposure.
Multiple Isoforms or Splice Variants
The target protein may exist in several isoforms or splice variants with slightly different molecular weights. These can produce multiple bands that remain detectable across sequential probing steps.
Cross-Reactivity with Homologous Proteins
Antibodies may recognize off-target proteins that share similar epitopes or structural features with the intended target, leading to unintended bands during the second detection.
Insufficient Antibody Specificity
The primary antibody may exhibit cross-reactivity with unrelated proteins, especially in complex lysates, causing multiple bands regardless of the stripping step.
To elucidate the origin of the observed bands, several strategies can be employed:
Assess Antibody Specificity
Perform Western blotting using well-characterized samples known to express the target protein. The presence of multiple bands in such controls may indicate non-specific binding or recognition of isoforms.
Validate with Alternative Antibodies
Use different primary antibodies targeting distinct epitopes of the protein. Consistent banding patterns may support the existence of splice variants or confirm antibody specificity.
Employ Mass Spectrometry
For definitive identification, excise bands of interest and analyze them using mass spectrometry to determine their protein composition and assess potential homology.
If these strategies do not resolve the issue, further optimization of experimental parameters, such as blocking conditions, antibody dilution, and membrane handling, should be considered.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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