Anti phospho akt ser473 polyclonal antibody

Purified recombinant GST-GAPDH or GST-GAPDH (Pro²³⁴Ser) or GST (5 μg) were preabsorbed to 20 μl glutathione Sepharose 4B for 1 h at room temperature by rotation, collected by centrifugation at 1700×g for min, and then washed 3X with TBS and 1% Triton X-100 to remove any unbound protein. The beads were resuspended in 50 mM Tris (pH 7.5), 5 mM MgCl₂, 100 mM NaCl, 10 μm GTPγS (ICN Biochemicals Inc., Irvine, CA), and then 5 μl rat liver cytosol added (~20 μg of total protein and depleted of GAPDH) [29 ] and incubated for an additional 2 h at RT. The beads were washed 4X with 1 ml of 50 mM Tris (pH 7.5), 10 mM MgCl₂, and 100 mM NaCl, and the beads with bound proteins boiled in sample buffer, separated by SDS-PAGE, and then transferred to nitrocellulose. The blot was blocked as above, and the membrane probed with an anti-GAPDH monoclonal antibody (Chemicon Intl.), anti-GST monoclonal (Novagen, Madison, WI), anti-phosphoAkt (Ser473) polyclonal antibody (Cell Signaling), or with anti-Akt 9 (pan) polyclonal antibody (Cell Signaling), washed, incubated with an HRP-conjugated secondary antibody, and then developed with ECL.