Protein g sepharose

To examine the changes in de novo synthesis of Gag upon Se treatment, the medium was replaced with methionine-free media containing Click-IT AHA (l-azidohomoalanine) (Life Technologies) (50 µM). Cells were lysed with 1% SDS, and the Click-IT reaction was carried out on 50 µg of protein lysate using a Click-IT Protein Reaction buffer kit (Life Technologies) and biotin-alkyne (40 µM). The labeled material was precleared with normal rabbit serum and 25 µl of a 50:50 slurry of protein G-Sepharose (Thermo Scientific) and incubated with anti-p24 antibody and a 50:50 slurry of protein G-Sepharose.

The membrane proteins were enriched by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific), following manufactory protocol. Briefly, HEK 293T cells transfected with wild type or L640R mutant Gpr56 cDNA were stimulated with collagen III or the vehicle (acetic acid) for 5 minutes, as described in previously [10] (link). About 5×10⁶ cells per sample were used for membrane protein extraction. The isolated membrane fraction samples were precleared for 1 h with protein G Sepharose (Invitrogen), followed by incubation with rabbit anti-GPR56^C (199) antibodies along with protein G-Sepharose. The immune complexes were washed and eluted with Laemmli Buffer for western blot. GPR56^N and GPR56^C proteins were detected by mouse anti-GPR56^N (H11) and rabbit anti-GPR56C (199), respectively.

U373-MG cells were starved of methionine and radiolabeled for 5–10 min with 100 μCi/ml of [³⁵S] Met. Following radiolabeling, cells were either chased with 2 mM methionine-supplemented media or immediately prepared for lysis as follows. Cells were washed twice with chilled PBS and lysed in RIPA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 10 mM iodoacetamide, and 1x proteasome inhibitor cocktail (P8340, Sigma Aldrich)). The lysates were rocked for 30 min then centrifuged for 10 min at 11,000 rpm to remove nuclei and insoluble material. Supernatant fractions were treated with 10 μg w6/32, 10 μg HC10, and 10 μg HCA2 to isolate MHC class I and with 20 μl anti-calnexin antiserum as a radiolabeling and immunoisolation recovery control. After 2 h a mix of protein A-Sepharose (GE Healthcare) and protein G-Sepharose (Life Technologies) beads was added and incubated for 1 h. Beads were washed four times with 0.5% Nonidet P-40, 10 mM Hepes pH 7.4, 150 mM NaCl and then proteins were eluted, separated by SDS-PAGE under reducing conditions and visualized by fluorography. For inhibition of proteasomal activity, cells were pre-treated for 60 min with 25 μM lactacystin (Abcam), starved of methionine in the presence of 25 μM MG132 (Boston Biochem), radiolabeled in the presence of both inhibitors, and chased in the presence of 25 μM MG132.