Methionine

Ab in synovial fluid was screened by immunoprecipitation (IPP) using radiolabeled HeLa cell extracts as previously described [17 (link)]. Briefly, 1 × 10⁷ HeLa cells were labeled with 9.25 MBq of [³⁵S]-methionine (PerkinElmer, Waltham, MA, USA) in 10 ml of methionine-free minimal essential medium and incubated at 37 °C for 18 h. The labeled cells were washed three times with phosphate-buffered saline (137 mmol/l NaCl, 8.1 mmol/l Na₂HPO₄, 2.68 mmol/l KCl, 1.47 mmol/l KH₂PO₄) and sonicated in IPP buffer (10 mM Tris–HCl, 500 mM NaCl, 0.1% Nonidet P-40). The soluble supernatant in IPP buffer was collected by centrifugation at 10,000 g for 10 min. Two microliters of synovial fluid was bound to 2 mg of Protein A CL-4B Sepharose beads (GE Healthcare, Uppsala, Sweden) in IPP buffer for 2 h at room temperature under constant rotation. After washing four times with IPP buffer, IgG-coated Sepharose beads were mixed with the [³⁵S]-methionine-labeled HeLa cell extracts for 2 h at 4 °C. After washing four times with IPP buffer, the IgG-coated beads were resuspended in sodium dodecyl sulfate (SDS) sample buffer and heated for 5 min at 95 °C. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by radiography.

5 ml of exponentially growing cultures of WT, ltv1Δ and yar1Δ [PSI⁺] cells were grown in presence of radiolabeled [³⁵S] methionine (PerkinElmer Life Sciences) for 10 min, and then chased in medium containing 50 mM cold methionine for 15 min before harvesting. Protein extracts were prepared as described above and analyzed by 2D-gel electrophoresis followed by autoradiography.