Western blotting is a technique that separates proteins based on size. In general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:
- Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein
- Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases.
- Splice variants and isoforms - alternative splicing and isoforms may create different sized proteins produced from the same gene.
- Relative charge - the composition of amino acids (charged vs non-charged).
- Multimers - e.g. dimerization of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands.