Chunk #16 — Experimental — Stable Expression of the A118/N40-hMOPR or G118/D40-hMOPR in CHO Cells — Immunoprecipitation of 35S-Labeled G118/D40- or A118/N40-hMOPR
At each chase time point, medium was aspirated and cells in two wells of 6-well plates were briefly washed and detached with 10 mM phosphate buffer/1 mM EDTA/1 mM glucose. Two wells of labeled cells were then collected in 1.5 ml microfuge tubes, pelleted by centrifugation at 1,000 g, and stored at -80°C until further experiments. Cells were solubilized with 400 μl of TTSEC buffer, end-to-end mixed for 30 min at 4°C, and centrifuged at 13,500 g for 10 min. Supernatants were filtered through 0.2-μm spin cartridges. Immunoprecipitation was performed twice in tandem with HA antibody (HA.11) followed by PANSORBIN and centrifugation to purify the 35S-labeled receptor for satisfactory signal/noise.
