Ampkα rabbit monoclonal antibody

The hypothalamus was homogenized in the lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) with a cocktail of protease inhibitors (Sigma Aldrich). Proteins (20 or 40 μg/lane) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with AMPKα rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), pAMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology; dil 1:1000), IKK-β rabbit monoclonal antibody (Abcam; dil 1:500) or α-tubulin mouse antibody (Sigma Aldrich; dil 1:1000) overnight at 4°C and then with secondary antibody against rabbit or mouse IgG (Promega; dil 1:2500) for 1 h at RT. The signals were visualized with the ECL system (Pierce). The expression level of α-tubulin was used to normalize the data.

The hypothalamus was homogenized in the lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 12% glycerol, 0.5 mM DTT, 0.1 mM EGTA) with a cocktail of protease inhibitors (Sigma-Aldrich). Separation of proteins and Western blot analyses were performed as previously described (Chun et al., 2004 (link)). Briefly, proteins (20 or 40 μg/lane) were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with AMPKα rabbit monoclonal antibody (Cell Signaling Technology; 1:1000), pAMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology; 1:1000), and α-tubulin mouse antibody (Sigma-Aldrich; 1:1000) overnight at 4°C and then with secondary antibody against rabbit or mouse IgG (Promega; 1:2500) for 1 h at RT. The signals were visualized with the ECL system (Pierce). The expression level of α-tubulin was used to normalize the data.