Cells were starved for 20 min in methionine-free, cysteine-free medium, labelled with [35S]methionine/[35S]cysteine) (Perkin-Elmer) for 30 min and then chased in medium containing excess cold methionine and cysteine (Sigma-Aldrich) at 37°C. Samples taken at the indicated time-points were lysed in 1% NP-40/TBS pH 7.6 with Roche complete protease inhibitor and 10mM iodoacetamide (IAA). Post-nuclear supernatants were pre-cleared with a combination of IgG-sepharose and Protein G Dynabeads for 1 to 2 h on a rotator at 4°C, and then incubated (rotating) with a PD-L1-specific antibody (AF156, R & D systems) for 1 h before adding Protein G Dynabeads for a further 2 h at 4°C. After 4 washes in 0.2% lysis buffer, samples were eluted in SDS sample buffer for 10 min at 70°C. Where indicated, eluates were treated with Endoglycosidase H (New England Biolabs) according to the manufacturer’s instructions before separation by SDS-PAGE. Gels were fixed, dried at 80°C for 2 h and processed for autoradiography. Phosphor screens were exposed to the radiolabelled proteins in fixed, dried gels overnight. Screens were scanned using a TYPHOON scanner and bands quantified using ImageJ.
35s methionine 35s cysteine
Manufactured by PerkinElmer
Sourced in United States
[35S]methionine/[35S]cysteine is a radioactive labeling reagent containing the isotopes sulfur-35 ([35S]) bound to the amino acids methionine and cysteine. It is commonly used in various biochemical and cell biology applications to study protein synthesis, localization, and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Methionine and cysteine, by Merck Group (1 mentions)
Methionine, by PerkinElmer (1 mentions)
Cysteine, by PerkinElmer (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Endoglycosidase h, by New England Biolabs (1 mentions)
Typhoon system, by GE Healthcare (1 mentions)
Fla 7000 phosphorimager, by Fujifilm (1 mentions)
Anti flag m2 antibody beads, by Merck Group (1 mentions)
4 protocols using 35s methionine 35s cysteine
1
Pulse-chase analysis of PD-L1 maturation
2
Assessing Mitochondrial Protein Translation
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The translation of proteins encoded by the mtDNA in patient fibroblasts was assessed by labelling with 35S-methionine/35S-cysteine (Perkin Elmer) as described previously [41] . Cytosolic translation was inhibited by co-incubation of the radioisotopes with 100 µg/ml emetine dihydrochloride. Total cell protein was extracted from both control and patient fibroblasts and 50 µg loaded onto a 15%–20% gradient gel for SDS-PAGE. Assessment of protein loading was achieved by Coomassie blue staining, and the gel was visualised by exposure to a blank PhosphorImager screen that was imaged using a Typhoon system (GE Healthcare).
3
Pulse-chase analysis of protein dynamics
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Pulse-chase experiments were performed as described previously [13 (link)]. In brief, cells were labeled for 30 min at 37 °C in cysteine/methionine-free DMEM supplemented with 5% dialyzed FBS and 0.1 mCi/mL (35S)cysteine/(35S)methionine (PerkinElmer, Waltham, MA, USA). After washing with ice-cold PBS, cells were chased in normal culture medium at 37 °C. Cells were lysed in TNE buffer and then subjected to immunoprecipitation with anti-FLAG M2 antibody beads (Sigma-Aldrich). Immunoprecipitates were analyzed by SDS–PAGE and autoradiography using a FLA7000 phosphorimager (Fujifilm, Tokyo, Japan).
4
Visualizing VEGF-C Protein Secretion
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293 T cells were transfected with plasmids encoding full-length VEGF-C, VEGF-C-ΔC, VEGF-C-CT, both VEGF-C-ΔC and VEGF-C-CT, or with an empty plasmid and metabolically labeled with [35S]-cysteine/[35S]-methionine (PerkinElmer, Waltham, MA). After 48 hours, supernatants were harvested and immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography.
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