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Why does the observed western blot band size (i.e., molecular weight) differ from the expected band size for some proteins?

Typically, SDS-PAGE allows electrophoretic separation of proteins in a sample by molecular weight (MW), with larger proteins migrating more slowly than smaller proteins. Loading protein markers of established size (e.g., Trident Prestained Protein Ladders) allows researchers to approximate the MW of their target protein. In the simplest scenario, the MW of a protein is determined by its amino acid constituents. However, there are various factors that can affect protein migration on SDS-PAGE, resulting in an observed signal that differs from the expected MW.

These factors include:

  1. Protein post-translational modifications (PTM):
    1. Sumoylation: Sumoylation of the target protein can cause a ~15-17 kDa increase of the observed MW due to the covalently attached SUMO protein.
    2. Ubiquitination: Addition of a single ubiquitin (8.5kDa), or a chain of ubiquitin proteins, will increase the target protein’s observed MW.
    3. Glycosylation: Glycosylation will increase the size of the target protein. Treating cells with glycosylation inhibitors (e.g., tunicamycin to block N-linked glycosylation or Benzyl-α-GalNAc (BαG) to block O-glycosylation) can be used to generate the unmodified target protein.
    4. Phosphorylation: Phosphorylation will increase the observed size of the target protein due to the negatively charged phosphate group impacting SDS binding, thus altering the protein’s electrophoretic mobility.
    5. Protein post-translational cleavage: Many proteins are synthesized as pro-proteins, and then cleaved to give the active/mature forms with decreased MW (e.g., pro-caspases to active caspases).
    6. Phosphatidylethanolamine (PE) conjugation: PE conjugation of proteins can lead to a decrease of observed target protein size (e.g., conversion of LC3-I to LC3-II via PE- conjugation).
  2. mRNA splice variants (isoforms):
    Alternative splicing enables an mRNA to synthesize differently sized protein variants (isoforms) from the same gene that may have different cellular functions or properties.
  3. Protein multimers:
    Target proteins may form multimers (dimers, trimers, and so on) and display one or more bands of increased MW. Use of reducing conditions with SDS-PAGE can decrease the extent of multimerization.
  4. Protein charge:
    The observed size can also be influenced by the amino acid composition of the target protein.