For 35S-methionine/cysteine labelling, HEK293E cells were seeded in six-well plates, serum starved for 16 h and then deprived of methionine and cysteine for an additional 2 h using DMEM without the above amino acids (Gibco). Cells were then treated with 10% dialysed serum containing 10 μCi ml−1 of 35S-Met/Cys (Perkin Elmer) and 250 nM of torin1 were indicated. Cells were lysed in RIPA buffer without SDS and 10 μl of the protein extract measured by LS6500 Multi Purpose Scintillation Counter (Beckman Coulter). Experiments were performed in independent triplicates (n=3) each of which was performed in a technical duplicate.
35s met cys
Manufactured by PerkinElmer
Sourced in United States
35S-Met/Cys is a radioisotope-labeled amino acid mixture containing L-[35S]methionine and L-[35S]cysteine. It is a widely used tool for metabolic labeling and protein synthesis studies in cell and molecular biology research.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Rpmi medium, by Thermo Fisher Scientific (3 mentions)
Personal molecular imager, by Bio-Rad (2 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ls 6500 multi purpose scintillation counter, by Beckman Coulter (1 mentions)
Scintillation counter, by Beckman Coulter (1 mentions)
Typhoon 9400, by GE Healthcare (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Sp6 polymerase, by New England Biolabs (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Dmem culture media, by Corning (1 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Protein concentration assay, by Bio-Rad (1 mentions)
Dmem cys met, by Merck Group (1 mentions)
Complete inhibitor cocktail, by Roche (1 mentions)
30 protocols using 35s met cys
1
Metabolic Labeling of Proteins with 35S
2
Measuring Cellular Protein Synthesis
3
Radiolabeling of Mouse Adipocytes
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Mouse adipocytes (0.25 × 106 cells) were pre-incubated in methionine-free RPMI medium (Invitrogen) with 10% FBS for 30 min. [35S]Met/Cys (250 μCi, Perkin-Elmer) was added and incubated at 37 °C with 5% CO2 for 15 min. Labeled cells were lysed in RIPA buffer (Thermo Fisher) and analyzed by Tris-glycine SDS-PAGE, fixation and autoradiography.
4
Pulse Labeling of Mitochondrial Proteins
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A 30-min pulse labeling of mitochondrial nascent chains with 35S Met-Cys (EasyTag-PerkinElmer) in cultured cells was performed as described (48 (link)). All samples were treated with Benzonase according to the manufacturer’s instructions and then mixed with gel loading buffer [186 mM tris-HCl (pH 6.7), 15% glycerol, 2% SDS, bromophenol blue (0.5 mg/ml), and 6% β-mercaptoethanol]. A 12 to 20% gradient SDS-PAGE was used to separate samples, then dried for exposure with a Phosphoscreen, and scanned with a Typhoon 9400 (GE Healthcare) for quantification. Gels were rehydrated in water and Coomassie-stained to confirm loading.
5
Radiolabeling of Virus-Infected Cells
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Confluent human A549 and chicken DF-1 cells in a 24-well plate were either left uninfected or infected with the viruses at an MOI of 2 (A549) or MOI of 1 (DF-1) for 1 h and incubated in DMEM containing 0.15% BSA. At various times after infection, cells were washed and cultured in methionine, cysteine-free DMEM media (Gibco) for 30 min. Then, cells were labeled with 35S-Met/Cys (25 μCi, PerkinElmer) in the same medium for 30 min at 37°C. Cells were lysed with 50 ul of NP-40 lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol), and 15 ul of the radiolabeled lysates were resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. The dried gels were exposed on a phosphor screen and visualized using Personal Molecular Imager (Bio-Rad). Volume analysis was performed using Quantity One 1-D analysis software (Bio-Rad).
6
Metabolic Labeling of Viral Proteins
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2 hours post-infection (hpi) the media was replaced with Met/Cys-free media (Gibco). At 2.5 hpi 10 μCi/mL [35S]-Met/Cys (Perkin Elmer) was added. For EC optimisation, GuHCl (2 mM final concentration) was added to media at 3, 3.5, or 4 hpi.
7
Affinity Purification and Radiolabeling of Proteins
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Frozen or fresh cultures of staphylococci were washed and blocked with ice-cold 4% PBSB. The target protein or lysate was added in ice-cold 4% PBSB or lysis buffer, and incubated at 4 °C for 60 min. After washing thrice by three minutes centrifugation at 6000 rpm and 4 °C, bound proteins were eluted with 0.2 M glycine (pH 2.2) and neutralized by addition of 1 M Tris (pH 9.1) or with 1% SDS. Radiolabeling was performed and the lysates prepared as recently reported [72 (link)]. Briefly, cells were labeled at 37 °C using methionine- and cysteine-free medium supplemented with 10 mCi/mL [35S]Met/Cys (PerkinElmer, Waltham, MA, USA).
8
Transcription and Translation of Genes
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All genes of interest were transcribed by means of SP6 polymerase (NEB, England) according to the manufacturer’s instructions. RNA was snap frozen in liquid nitrogen and stored at −80 °C. Translation war performed with the Flexi Retic Kit (Promega, Mannheim, Germany) for 90 min in the presence of 35S Met/Cys (Perkin Elmer, Waltham, MA, USA). Translated preproteins were aliquoted, snap frozen, and stored at –80 °C until further use.
9
Protein Synthesis Regulation by Arsenite
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Semiconfluent cell monolayers were first starved in medium lacking methionine and cysteine for 30 min and then treated with the indicated arsenite concentrations for 30 min with or without ISRIB. Subsequently, newly synthesized proteins were labeled with 50 µCi/mL 35S Met/Cys (Perkin-Elmer) for another 90 min. Cells were then lysed, and proteins were separated using SDS/PAGE. Subsequently, gels were dried on Whatman paper and analyzed using a phosphor imager.
10
Pulse Labeling of Proteins for Immunoprecipitation
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Exponentially growing cells in SD medium containing essential amino acids were harvested and resuspended in the same medium supplemented with 0.15 mCi of 35S-Met/Cys (PerkinElmer) for pulse labeling. Cells were then pelleted and washed with cold H2O to remove residual medium containing 35S-Met/Cys. Labeled cells were lysed in 2× SDS buffer. Supernatants were recovered by centrifugation and diluted 20-fold with buffer A (150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Na–HEPES, pH 7.5, and 1% Triton X-100) before being subjected to immunoprecipitation (IP) with anti-ha antibody. The precipitated proteins were separated by SDS-PAGE, followed by autoradiography. The volumes of supernatants applied to IP were adjusted to equalize the amounts of 35S incorporated into total proteins using the trichloroacetic acid (TCA) precipitation assay as previously described (47 (link)).
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