All lanes : Anti-Cripto1 antibody - Embryonic Stem Cell Marker ( ab19917 ) at 1 µg/ml Lane 1 : Human Embryonic Stem Cell Lysate at 20 µg Lane 2 : Cripto protein at 0.1 µg Lane 3 : Human Embryonic Stem Cell Lysate at 20 µg with Human Cripto1 peptide (ab23310) at 1 µg/ml Lane 4 : Cripto protein at 0.1 µg with Human Cripto1 peptide (ab23310) at 1 µg/ml Secondary Rabbit IgG secondary antibody ( ab28446 ) at 1/10000 dilution Predicted band size : 21 kDa Observed band size : 17,17-21 kDa ( why is the actual band size different from the predicted? )