Af954

DPP-4 was immunodepleted from obese mouse plasma using bead-immobilized anti-DPP-4 antibody (R&D Systems, AF954) as previously described^{37 (link)}. In brief, 10 µg of anti-DPP-4 antibody was conjugated with 1.5 mg of Protein-G Dynabeads (Invitrogen, 10004D) in 200 µl of binding buffer. After immobilization of anti-DPP4 antibody to beads, 40 µl of DIO mouse plasma and 60 µl of PBS were mixed with 40 µl of antibody-coated bead suspension. The resultant mixture was incubated for 2.5 h at 4 °C on a rotating wheel. After 2.5 h, Dynabeads were separated with a magnet and discarded. This was followed by one more round of immunocapture of DPP4 and discarding of beads. DPP-4 proteolytic activity assay was performed on immunodepleted plasma to confirm DPP-4 immunodepletion. For some experiments, DIO mouse plasma was incubated with anti-DPP4-conjugated or control IgG-conjugated beads, followed by elution of the bound material using 50 mM glycine buffer, pH 2.8.