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.