Mouse anti myc monoclonal antibody

Polyclonal rabbit anti-phospho-Tyr15 Cdc2, mouse monoclonal Cdc2, mouse monoclonal cyclin B1, anti-HA rabbit polyclonal antibody, anti-Myc mouse monoclonal antibody, rabbit polyclonal anti-β–actin, HRP conjugated mouse and rabbit polyclonal antibodies were all purchased from Santa Cruz Biotechnology (USA), IMGENEX Corporation (USA) or Cell Signaling Technology, Inc. (USA).

HEK 293T cells in a 10-cm Petri dish were transfected with myc-FTL, flag-Gadd45a, and their control expression vectors in different combinations using the Attractene Transfection Reagent (Qiagen). Forty-eight hours after transfection, cells were rinsed twice with PBS and lysed in 1 ml lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.5% Nonidet P-40 with proteinase and phosphatase inhibitors. The cell lysates were briefly centrifuged to remove cell debris, and the supernatant was separated into three aliquots (300 μl per aliquot). The first aliquot was incubated with 10 μl of mouse anti-myc monoclonal antibody (Santa Cruz Biotechnology: sc-40), the second aliquot was incubated with 2 μl of mouse anti-flag monoclonal antibody (MBL, Nagoya, Japan: M185-3L), and the third aliquot was incubated with 2 μl of mouse monoclonal anti-IgG antibody (Abcam: Ab18413). After incubation at 4°C overnight, the immune complex was precipitated with 20 μl Protein G Plus-Agarose (Santa Cruz Biotechnology: sc-2002). The immunoprecipitates were washed four times with lysis buffer, and then subjected to electrophoresis. The antibodies used for western blotting were rabbit anti-myc antibody (Santa Cruz Biotechnology: sc-789) and rabbit anti-flag antibody (Sigma-Aldrich: F7425).