Glut4 agarose antibody

Human nondiabetic and diabetic pre-adipocytes were grown to confluency and differentiated for 4 days in differentiation media. Cells were harvested and 200 µg in 200 μL volume of lysate was used for coimmunoprecipitation assay. Lysates were rocked with Protein A/G Agarose (Santa Cruz sc2003, Dallas, TX, USA) for 30 min at 4 °C to clear nonspecific binding then centrifuged at 2000 rpm for 1 min to remove protein beads. Immunoprecipitation was performed using 2 μg Glut4-agarose antibody (Santa Cruz sc53566 AC, Dallas TX, USA) or IgG (Santa Cruz sc-51993, Dallas, TX, USA) as control and rocked overnight at 4 °C. Lysate was then centrifuged, and pellet was washed with PBS and resuspended in Laemmli buffer. Western blot analysis was performed as described above.

3T3-L1 adipocytes were harvested and 200 µL of 1 μg/μL of lysate was used for coimmunoprecipitation assay. Lysates were rocked with Protein A/G Agarose (Santa Cruz sc2003) for 30 min at 4 °C to clear nonspecific binding and centrifuged at 2000rpm for 1 min. The supernatant weas rocked with 2 μg Glut4-agarose antibody (Santa Cruz sc53566 AC) overnight at 4 °C and pellet was washed and resuspended in Laemmli buffer followed by western blot analysis as described above.