Why Does Alix Protein Antibody Show Two Bands in WB? Which One Is Correct
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Reviewing Literature and Databases: Investigate reported molecular weights of Alix protein, known isoforms, and documented modifications.
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Using Molecular Weight Markers: Include molecular weight markers in WB to estimate the molecular weight of the detected protein.
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Analyzing Protein Degradation or Phosphorylation: If degradation or phosphorylation is suspected, validation can be performed by adjusting sample preparation methods (e.g., using protease inhibitors) or employing phosphorylation-specific antibodies.
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Verifying with Alternative Antibodies: Conduct WB using another antibody against Alix protein to assess whether both bands are consistently detected.
Several factors may contribute to the appearance of two bands in Western Blot (WB) when using an Alix protein antibody:
1. Protein Isoforms
Alix protein may exist in different isoforms, which differ slightly in molecular weight, leading to distinct bands on the gel.
2. Protein Degradation Products
Partial degradation of Alix protein during cell lysis may result in the formation of smaller degradation fragments. These fragments can also be recognized by the same antibody, appearing as lower molecular weight bands in WB.
3. Phosphorylation or Other Protein Modifications
Post-translational modifications, such as phosphorylation, may induce subtle shifts in molecular weight. These modifications can alter the protein’s migration in SDS-PAGE, leading to multiple bands.
To accurately identify the target band, consider the following approaches:
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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