Ab21635

Magnetic Beads Protein G were coated with 5 mg of primary antibody in RIP wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM MgCl 2 , and 0.05% NP-40) containing 5% BSA overnight with rotation at 4 C. HEK293 cells expressing HA-MS2P, with Gria1 and Gria2 and Gria2I-MS2 or AK045738-MS2 were collected in 100 mL of RIP lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, 1 mM PMSF/cocktail) and 10 mL of RNase inhibitor (Ambion, AM2694), and incubated on ice for 10 min. The lysates were then centrifuged at 14,000 rpm for 10 min at 4 C and the nuclear fraction was collected and added with 900 mL of beads-antibody complex in RIP Buffer containing 860 mL RIP wash buffer, 35 mL 0.5 M EDTA, and 5 mL RNase inhibitor, and incubated with rotation overnight at 4 C. 5 mL of the RIP lysate without IP was used as input. After washing, the precipitates were divided into two parts; one was mixed with 5 mL of western blotting sample buffer and incubated for 5 min in boiling water; this blot was used to detect REST/NRSF (1: 1000, Abcam, ab21635). Another was treated with proteinase K at 55 C for 30 min with shaking to digest the protein, followed by DNA extraction and probed with Gria2.