All lanes : Anti-Chk2 (phospho T26) antibody ( ab26338 ) at 1 µg/ml Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg Lane 2 : Gamma-Irradiated HeLa Lysate - 4 hour exposure at 20 µg Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Human Chk2 (phospho T26) peptide ( ab27902 ) at 1 µg/ml Lane 4 : Gamma-Irradiated HeLa Lysate - 4 hour exposure at 20 µg with Human Chk2 (phospho T26) peptide ( ab27902 ) at 1 µg/ml Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Human Chk2 peptide (ab37237) at 1 µg/ml Lane 6 : Gamma-Irradiated HeLa Lysate - 4 hour exposure at 20 µg with Human Chk2 peptide (ab37237) at 1 µg/ml Secondary Lanes 2 - 6 : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution Performed under reducing conditions. Predicted band size : 61 kDa Observed band size : 75 kDa ( why is the actual band size different from the predicted? )