35s methionine cysteine

Vero E6 cells were infected with either MP-12 or its mutants at a multiplicity of infection (m.o.i.) of 3. At 16 h post infection (p.i.), the culture media was replaced with methionine/cysteine-free medium. After starvation for 30 min, the infected cells were labeled with 100 μCi/ml of ³⁵S-methionine/cysteine (1,000 Ci/mmol; MP Biomedicals) for 1 h. The radiolabeled cells were suspended in 2x SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by Coomassie Blue staining or autoradiography.

MHV-infected 17Cl-1 cells were incubated in methionine-free medium for 30 min and then incubated in medium containing 50 μCi/mL [³⁵S]methionine/cysteine (1,000 Ci/mmol; MP Biomedicals) for 30 min. Whole-cell lysates were prepared in SDS sample buffer and analyzed on 12.5% SDS/PAGE.

Twenty hours after transfection, COS-1 cells transfected with PMT2-FVIII plasmids were metabolically labeled with [35S]-methionine/cysteine (250 μCi/mL in methionine/cysteine-free DMEM) (MP Biomedicals) for 45 minutes, followed by a 30-minute incubation in complete medium. Cell extracts were prepared by lysis in the NP-40 lysis buffer and immunoprecipitated with anti-FVIII antibody coupled to CL-4B sepharose. Immunoprecipitated proteins were washed with NP-40 lysis buffer and PBS, and resuspended in 50 mM Tris-Hcl pH 7.5, 150 mM NaCl, 2.5 mM CaCl₂ and 5% glycerol (buffer A). Immunoprecipitated FVIII in buffer A were divided into two aliquots for incubation in the absence or presence of 5 U/ml thrombin (Sigma) at 37 °C for 30 minutes. The resulting samples were separated in a 12% SDS-PAGE gel and visualized by exposing to an X-ray film using a Kodak Biomax Transcreen LE.