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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 http://www.hprd.org/
      2. ProSite http://www.expasy.org/prosite/
      3. ELM http://elm.eu.org/
      4. CBS data sets http://www.cbs.dtu.dk/databases/
      5. CBS prediction Servers http://www.cbs.dtu.dk/services/
  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.