Anti h2 db antibody 28 14 8

Brain lysates in Lysis Buffer (150 mM NaCl, 50 mM Tris, 0.25 % sodium deoxycholate, 1% NP-40, 1 mM EGTA, 1 mM PMSF, 1X Pefabloc (Roche)) were prepared by shearing ten times in a dounce homogenizer, then centrifuging at 12,000g for 10 minutes. Supernatants were precleared by incubation with Protein G-agarose beads (Invitrogen), and protein amounts were measured using a BioRad protein assay. Normalized lysates were then incubated with 12 μg anti-H2-Db antibody 28-14-8 (BD Biosciences, #553600) overnight at 4°C. Protein G-agarose was incubated with samples for 45 minutes. Beads were washed three times with lysis buffer, and then heated to 85°C for 3 minutes in NuPAGE LDS 4X Sample Buffer (Invitrogen) + 1% β-2 mercaptoethanol. Samples were then electrophoresed on an SDS-PAGE gel, transferred to Immobilon-P PVDF transfer membrane (Millipore), and Western blotted with rabbit monoclonal antibodies to H2-Db, made against the extracellular domain of H2-Db.