Anti-Cyclin B1 pS126 Antibody - Western Blot. Western blot of Affinity Purified anti-Cyclin B1 pS126 antibody shows detection of a band ~48 kD corresponding to phosphorylated human Cyclin B1 (arrowheads) in various whole cell lysates. Lysates tested were lane 1 - HeLa (cervical carcinoma), lane 2 - H23 (lung carcinoma), lane 3 - Hep3b (Hepatocarcinoma), lane 4 - T98G (Glioblastoma) and lane 5 - Daudi (B cell lymphoblast). Each lane contains approximately 50 ug of lysates, separated by 12% SDS-PAGE using a 5% stack run at 100 volts until the dye front cleared the bottom of the gel. Transfer occurred overnight at 4C at 15 mAmps. The membrane was blocked with 5% non-fat dry milk in TTBS for 1 h at room temperature followed by addition of a 1:100 dilution of the antibody allowed to react for 2h at room temperature. After washes with TTBS a 1:5000 dilution of HRP conjugated Gt-a-Rabbit IgG [H&L] MX (LS-C60865) was added for 1 h at room temperature. After additional washes the membrane was incubated with ECL mix 1:1 for ~3 min. Excess detection solution was drained off and the membrane was exposed to Kodak film Xomat blue XB-1 for about 20 sec. Other detection systems will yield similar results. Personal Communication, Luca Cote, Temple U.