Labeling of viruses with [35S]methionine-cysteine (PerkinElmer) was carried out as described previously, with slight modification (25 (link)). Briefly, confluent monolayers of cells were infected with each individual virus at a multiplicity of infection (MOI) of 0.1 PFU/cell for 4 h at 37°C. The medium was replaced with RPMI 1640 lacking methionine and cysteine (Sigma-Aldrich). Cells were starved for 2 h and then supplemented with 1 Mbq of [35S]methionine-cysteine (PerkinElmer) ml−1. At 72 h following virus infection, each labeled virus was purified by cesium chloride (CsCl) density gradient centrifugation as described below.
35s methionine cysteine
Manufactured by PerkinElmer
Sourced in United States, Germany
[35S]methionine/cysteine is a radioactive amino acid labeling reagent used in various biochemical applications, such as protein synthesis studies and protein detection. It provides a means to incorporate radioactive sulfur-35 into proteins, allowing for their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pngase f, by New England Biolabs (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
L cysteine, by Thermo Fisher Scientific (2 mentions)
Anti ha agarose beads, by Merck Group (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
L methionine, by Thermo Fisher Scientific (2 mentions)
Native imagequant software, by GE Healthcare (2 mentions)
Rpmi 1640, by MP Biomedicals (2 mentions)
Endoglycosidase h, by New England Biolabs (2 mentions)
Protein a agarose, by Thermo Fisher Scientific (2 mentions)
Typhoon fla 9000, by GE Healthcare (2 mentions)
Rpmi 1640 lacking methionine and cysteine, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Mouse anti p53 1c12, by Cell Signaling Technology (2 mentions)
Glutathione sepharose beads, by Macherey-Nagel (2 mentions)
Anti ppm1d, by Santa Cruz Biotechnology (2 mentions)
Anti histidine, by Santa Cruz Biotechnology (2 mentions)
Ppm1d inhibitor cct007093, by Merck Group (2 mentions)
49 protocols using 35s methionine cysteine
1
Radiolabeling and Purification of Viruses
2
Radiolabeling Shank3 Dynamics
3
Protein Stability Assay Protocol
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For protein stability assays, cells were incubated in methionine- and cysteine-free DMEM media containing 10% dialyzed FBS, and 55 μCi of [35S] methionine-cysteine (Perkin-Elmer, Boston, MA). Cells were then washed twice with PBS and incubated in chase medium (standard DMEM containing 10% FBS, 100 μg/ml methionine and 100 μg/ml cysteine) for up to 24 hours. For immunoprecipitation, equal amounts of cell lysates were incubated with the mouse anti-HA antibody overnight at 4°C, followed by incubation with protein A/G-sepharose (GE Healthcare, Piscataway, NJ) for 4 hrs. Beads were washed three times in RIPA buffer and proteins were eluted with 50μl of 3X Laemmli sample buffer. The radiolabeled proteins were separated by electrophoresis using 7.5% SDS polyacrylamide gels. The gels were then fixed, impregnated with Amplify (GE Healthcare, Piscataway, NJ), dried and exposed to film. Densitometric analysis was performed using NIH ImageJ software (NIH, Bethesda, MD).
4
Metabolic Radiolabeling and Immunoprecipitation
5
In vitro Transcription-Translation Assay with FMDV 3C Protease
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Coupled in vitro transcription and translation assays were performed using the TNT Quick Coupled Transcription/Translation system (Promega) as described previously (16 (link)). Reactions contained 10 μL lysate with 250 ng of pcDNA T7 expression plasmid and 0.5 μL [35S] methionine/cysteine (PerkinElmer). Reactions were incubated at 30°C for 40 min chasing with 2 μL of 50 mg/mL unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer. Samples were separated by SDS-PAGE before visualization of radiolabeled products by autoradiography.
For the trans-cleavage assays, the TNT reactions were supplemented with purified FMDV 3Cpro (a kind gift from Dr. Tobias Tuthill [57 (link)]) to the indicated final concentration from dilution of a 1 mM stock, simultaneous to the addition of unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer and the 3Cpro-mediated proteolysis of radiolabeled precursor monitored by SDS-PAGE.
For the trans-cleavage assays, the TNT reactions were supplemented with purified FMDV 3Cpro (a kind gift from Dr. Tobias Tuthill [57 (link)]) to the indicated final concentration from dilution of a 1 mM stock, simultaneous to the addition of unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer and the 3Cpro-mediated proteolysis of radiolabeled precursor monitored by SDS-PAGE.
6
Quantifying Intracellular Protein Synthesis
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Cells were washed twice with Phosphate-Buffered-Saline (PBS) and incubated for 1 min at 37 °C with labelling media (Met/Cys-free DMEM with the usual supplements, dialysed FBS and 120 Ci/mL of 35S-Methionine/Cysteine (Perkin Elmer)). Next, cycloheximide (C4859, Sigma-Aldrich) was added for 1 min at 37 °C at a final concentration of 100 μg/mL. Cells were then washed three times with cold PBS (plus 5 mM Met and Cys). They were then harvested in 0.5 mL of cold PBS and pelleted by centrifugation for 3 min at 3000 g at 4 °C. Cells were lysed for 30 min at 4 °C with NET buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% IGEPAL, with protease inhibitors) and, after centrifugation for 10 min at 10000 g, the supernatant was precipitated with cold 10% Trichloroacetic acid in order to measure the amount of intracellular radioactivity incorporated into protein. The percentage of intracellular radioactivity incorporated was calculated.
7
Analyzing Cell Surface Protein Trafficking
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Filter-grown cultures were maintained at confluence for 5 d at 37°C to ensure complete polarization, washed with DMEM lacking methionine or cysteine containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 5% dialyzed FBS, and incubated for 1 h at 37°C. Cultures were then pulse labeled with medium containing 300 μCi/ml [35S]methionine/cysteine (PerkinElmer, Waltham, MA) for 15 min at 37°C and chased in medium containing a 10-fold excess of methionine and cysteine at the indicated temperatures. Before processing, samples were placed on ice and cell surface biotinylation performed as described. After biotinylation, cells were solubilized in TEN-T lysis buffer (described earlier), and protein was immunoprecipitated with antibodies directed against E-cadherin or the Na,K-ATPase. Precipitated proteins were eluted in TEN (no Triton) with 1% SDS for 20 min at room temperature. Samples were then diluted 10-fold in TEN-T, and cell surface proteins were precipitated with streptavidin-conjugated beads (Pierce). Samples were analyzed by SDS–PAGE, fixed in 10% acetic acid/20% methanol and dried before exposure to x-ray film. Images were quantified using ImageJ.
8
Radioactive Protein Labeling and Immunoprecipitation
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Freshly cultured parasites were washed and resuspended in methionine-free RPMI 1640 (MP Biomedicals Inc., Aurora, OH). 200 µCi/mL of [35S] methionine/cysteine (PerkinElmer, Boston, MA) was added and parasites were incubated at 37°C for 2 h. Parasites were lysed in NETT buffer [10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100] using a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA)) and centrifuged to collect the supernatant. Lysates were pre-cleared with Protein G-Sepharose beads (GE Healthcare, Waukesha, WI) before antibody addition. Protein G-Sepharose beads were added and washed extensively with NETTS (10 mM Tris, pH 7.5, 500 mM, NaCl, 5 mM EDTA and 0.1% Triton X-100) and NETT buffers. Protein was eluted from the beads using elution buffer [10 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA], boiled and run on SDS-PAGE gel. Gels were fixed with fixing solution [25% Isopropanol alcohol; 10% Acetic Acid] for 30 min and enhanced with Amplify™ fluorographic solution (GE Healthcare, Waukesha, WI) for 45 min, dried under vacuum, and exposed to X-ray film for autoradiography.
9
In Vitro Fetuin-A and ApoM Synthesis
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Full-length V5-tagged fetuin-A and apoM mRNAs were in vitro transcribed from pcDNA3.1 vectors using the mMESSAGE mMACHINE kit (Ambion). mRNAs were utilized for in vitro translation in 100 μl reactions using methionine- and cysteine-free amino acid mix and nuclease-treated extracts from primary hepatocytes isolated from mice injected with Ad-shCtrl or Ad-shVig64 (link). Proteins were co-translationally radiolabeled by addition of 50 μCi [35S]-methionine/cysteine (PerkinElmer) to the reaction. V5-tagged protein products were immunoprecipitated with a V5-antibody conjugated to protein G Dynabeads (Life Technologies), washed 10 × with IP wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM KCl, 0.05% NP40, 0.5 mM DTT, 1 tablet cOmplete EDTA-free protease inhibitor cocktail (Roche) per 50 ml) separated by SDS–PAGE and visualized by autoradiography using x-ray films (Fuji).
10
In organello mitochondrial translation
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In organello translation assays were carried out in isolated heart mitochondria as described before (18 (link), 19 (link), 23 (link), 41 (link)). Heart or skeletal muscle mitochondria (500 μg) were incubated in 750 μl of translation buffer [100 mM mannitol, 10 mM succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KPi, 25 mM Hepes (pH 7.4), 5 mM ATP, 20 μM GTP, 6 mM creatine phosphate, creatine kinase (60 μg/ml), and all amino acids (60 μg/ml) except methionine and cysteine]. Mitochondria were supplemented with 150 μCi of [35S]methionine/cysteine (PerkinElmer) for 1 hour at 37°C. After labeling, mitochondria were washed in translation buffer and resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentration was measured, and 50 μg of mitochondrial protein was resolved by SDS-PAGE and visualized by autoradiography.
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