Infrared dye 800 conjugated anti rabbit igg

Individual hippocampi were sonicated in lysis buffer (10mM Tris, pH7.4, 100 mM NaCl, 1 mM EDTA, 1mM EGTA, 1mM NaF, 20 mM Na 4 P 2 O 7, 2mM Na 3 VO 4, 1% Triton X-100, 10 % glycerol, 0.1% SDS, and 0.5% deoxycholate) with Complete protease inhibitor cocktail (Roche, Indianapolis, IN) as we have previously published (14 (link)). Reducing agent (Thermo Scientific, Waltham, MA) and LDS (Thermo) were added to each sample. 20–30 μg of total protein was separated using NuPAGE 4–12% Bis-Tris gels (Thermo) and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) as previously described (13 (link)). Using Rockland (Pottstown, PA) Blocking Buffer for Fluorescent Western Blotting and the following antibodies, the blots were imaged and analyzed with Odyssey infrared scanning (LiCor Bioscience, Lincoln, NE) as previously described. Mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO) and used at 1:10,000. Primary antibodies purchased from Cell Signaling Technology (Danvers, MA) and used at 1:500 included: pS6K (Thr389, #9205), S6K (#9202), pAkt (Ser473, #9271), pAkt (Thr308, #9275), Akt (#9272), p-mTOR (Ser2448, #2971), mTOR (#2972), pAMPKα (Thr172, #2531), AMPKα (#2532). Secondary antibodies were Alexa Fluor 680 conjugated anti-mouse IgG (1:12,500, Thermo) and Infrared Dye 800 conjugated anti-rabbit IgG (1:12,500, Rockland).