Anti adiponectin antibody

Reduced and heat-denatured samples were separated on 10% SDS-polyacrylamide gels (acrylamide from National Diagnostics). For native gel electrophoresis, 5% polyacrylamide gels were prepared without SDS. Additionally, SDS and reducing agents were eliminated from the running and sample loading buffers. Following gel electrophoresis, proteins were transferred to nitrocellulose membranes in transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. Traditional immunoblotting procedures were followed, and results were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Thermo Scientific, Rockford, IL). Primary antibodies included: the anti-adiponectin antibody from Thermo Scientific (Rockford, IL); phospho-AMPKα (Thr172), phospho-Akt (Ser473), and MCP-1 antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA); and anti-MAPK (Erk 1/2) antibody from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Optical densities of all protein bands were analyzed using Image Studio Lite software (Licor Biosciences, Lincoln, NE).

Whole cell extracts were prepared by harvesting adipocyte monolayers in lysis buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Igepal CA-630, 1 mM PMSF, 1 mM pepstatin, 1ul aprotinin, 10 mM leupeptin, 1 mM 1, 10-phenanthroline, and 0.2 mM sodium vanadate. Adipocyte monolayers were scraped into the lysis buffer, subjected to one freeze-thaw cycle, and passed 3 times through a 20-gauge needle. The lysates were then centrifuged at 17, 500 x g for 10 minutes at 4°C. After removing the floating lipid layer, protein concentrations of the supernatants were determined by a BCA kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. As previously described [15 (link)], typical immunoblotting procedures were performed, and results were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Primary antibodies included anti-adiponectin antibody (Thermo Scientific, Rockford, IL), phospho-Akt Ser473, anti Akt, anti-acid ceramidase, anti-neutral ceramidase (Santa Cruz, Dallas, TX), anti β-Actin and anti-tubulin (Sigma Aldrich, St Louis MO). Anti-rabbit and anti-mouse IgG secondary antibodies were from Sigma Aldrich (St Louis, MO). Optical densities of all protein bands were determined using NIH image J software as previously described [16 ].