All lanes : Anti-ITCH/AIP4 antibody ( ab31097 ) at 1 µg/ml Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human ITCH/AIP4 peptide (ab32426) at 1 µg/ml Lysates/proteins at 10 µg per lane. Secondary IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution Performed under reducing conditions. Predicted band size : 103 kDa Observed band size : 96 kDa ( why is the actual band size different from the predicted? )