Cells were primed in medium containing DMEM, l -glutamine, pyruvate, nonessential amino acids, dialyzed serum (Life Technologies), and penicillin-streptomycin for 15 min at 37°C. Cells were metabolically labeled in medium containing 50 µCi [35S]methionine/cysteine (Hartmann Analytic). For the negative control, 100 µg/ml cycloheximide was added to the medium. Cells were collected at different time points, lysed in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, pH 8, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, and protease inhibitor cocktail. The proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (VWR International) that were then exposed to x-ray film.
35s methionine cysteine
Manufactured by Hartmann Analytic
[35S]methionine/cysteine is a radioisotope-labeled amino acid used in various biochemical and cell biology applications. It serves as a precursor for the metabolic labeling and detection of newly synthesized proteins.
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Lab products found in correlation
2 protocols using 35s methionine cysteine
1
Protein Synthesis Kinetics Protocol
2
Pulse-chase analysis of FGE variants
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HT1080 cell lines stably expressing FGE variants Ser155Pro, Gly247Arg and Ala279Val were either treated with 10 μM tazarotene and 20 μM bexarotene or DMSO (as control) for 3 days prior to the start of pulse‐chase experiments. Of note, the presence of the drug or DMSO was maintained in all supplemented media throughout the experiment. After starving for 1 h in a medium depleted of methionine and cysteine, the cells were pulsed with 35S‐methionine/cysteine (Hartmann Analytic) for 30 min. Cells and media were collected after incubation for various time points in unlabeled medium (chase). Cell lysis, FGE immunoprecipitation from cell lysate and media, SDS–PAGE and autoradiography, and image analysis using ImageJ software were as previously described (Schlotawa et al, 2018 (link)).
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