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Why is the observed Western Blot band size different from predicted size?

  1. Post-translational modification (PTM):
    1. Some post-translational modifications might lead to increased protein size, including phosphorylation, acetylation, methylation, glycosylation, sumoylation, ubiquitination, etc.
    2. Some post-translational modifications might lead to decreased protein size including phosphatidylethanolamine conjunction (e.g. LC3-II)
    3. Some proteins may be cleaved to form an active or mature form; this process will lead to a decreased protein size (e.g. Notch activation, Caspase activation, etc.)
    4. Some websites provide useful PTM information
      1. HPRD
      2. ProSite
      3. ELM
      4. CBS data sets
      5. CBS prediction Servers
  2. mRNA splice variants (Isoforms):
    Through alternative splicing, one gene can generate different proteins with different M.W. Regulation of alternative splicing depends upon cell type, conditions, etc.
  3. Multimerization:
    Some proteins could form dimers or multimers, increasing the M.W. This phenomenon usually can be found in reducing gel condition; however, strong interactions may still be seen with higher molecular weight proteins even in denaturing gel.
  4. Protein charge:
    The observed size could also potentially be influenced by the protein charge
  5. Different species
    Different species likely have different protein sequence and PTM, which can lead to a different protein M.W.