Why is the actual western blot band size different from the theoretical molecular prediction?
In Western Blotting analysis, the migration of proteins on the gel is based on their molecular weight. However, under certain circumstances, there is a discrepancy between the actual western blot band size and predicted molecular weight. The following factors are the common reasons for this. • 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 – alternative splicing 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.
