Immunoprecipitation of Glycogen Synthase was performed on HepG2 cells, glucose and serum starved for 36 hours. The antigen:antibody complex was formed by binding 500µg whole cell lysate with 5µg of mouse monoclonal antibody recognizing O-GlcNAc (Product # MA1-039) overnight on a rocking platform at 4°C. The immune-complex was then captured on 50µl Protein A/G Plus Agarose (Product #20463). The beads were washed to remove non-bound material and a low-pH elution buffer was used to dissociate bound antigen from the antibody-bound bead. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing Glycogen Synthase (Product # PA5-17459) at a dilution of 1:1000 overnight rotating at 4°C. Membranes were then washed in TBST and probed with Clean-blot IP detection reagent (Product #21230) at a dilution of 1:2000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Pico (Product #34087). The image is a Western blot with antibody PA5-17459 showing enrichment of glycogen synthase by O-GlcNAc precipitation. Lane 1 indicates 25 ug of HepG2 whole cell lysate input; Lane 2 indicates the IP elution.