Anti myc monoclonal antibody

The immunoblot analysis was essentially done by adopting a published protocol [7 (link)]. All recombinant yeast cells were grown in SC^Raf medium at 30°C till mid-log phase. The cultures were then diluted directly in induction media (SC^Gal). After 6 hours of induction, cultures were pelleted in a refrigerated centrifuge. Cells were lysed in 150μl of breaking buffer (50mM Tris-HCl (pH 7.5), 10% glycerol, 1% Triton X-100, 0.1% SDS, 150mM NaCl, 50mM NaF, 1mM Sodium Orthovandate, 50mM β-Glycerol phosphate, 5mM EDTA, 1mM phenylmethylsulfonylfluoride and the 1X protease inhibitor cocktail by vigorous vortex with 0.3-mm glass beads (Sigma)). Cell extracts were separated from glass beads and cell debris, collected in a new centrifuge tube by centrifugation, and further clarified by a 13,000 x g spin for 15 min at 4°C. The protein concentration of the supernatants was measured by BCA Protein assay kit (Pierce). Equal concentration of protein samples were fractionated by SDS-polyacrylamide gel electrophoresis using 12% polyacrylamide gels and transferred to Immobilon—P Transfer Membrane (Millipore). Membranes were probed with either anti-myc monoclonal antibody (Thermo Scientific) or anti-actin monoclonal antibody (Millipore). The primary antibody was detected using a horseradish peroxidase conjugated anti-rabbit antibody with the Millipore detection system.