Western blot All lanes: Rhombotin-1 antibody at 2 /mlLane 1: EC109 whole cell lysateLane 2: 293T whole cell lysate SecondaryGoat polyclonal to Rabbit IgG at 1/15000 dilution Predicted band size: 17 kDa Observed band size: 50kDa Why is the actual band size different from the predicted 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 and 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 - alternative splicing may create different sized proteins from the same gene · relative charge - the composition of amino acids (charged vs non-charged) multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands.