35s cys met

Manufactured by MP Biomedicals

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[35S]-Cys/Met is a radioactive labeled amino acid product containing cysteine and methionine labeled with the isotope sulfur-35. It is commonly used as a tracer in various biochemical and cell biology applications, such as protein synthesis studies and metabolic labeling experiments.

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Lab products found in correlation

Typhoon imager, by GE Healthcare (3 mentions) Phosphorimager plate, by GE Healthcare (2 mentions) Hepatozyme medium, by Thermo Fisher Scientific (1 mentions) Heparin, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Fibronectin, by Merck Group (1 mentions) Fumed silica, by Merck Group (1 mentions) Primaria dishes, by BD (1 mentions) William s e medium, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Nupage gel, by Thermo Fisher Scientific (1 mentions) Imagequant software, by GE Healthcare (1 mentions) X ray film, by GE Healthcare (1 mentions) M2 anti flag agarose beads, by Merck Group (1 mentions) Photographic film, by GE Healthcare (1 mentions)

7 protocols using 35s cys met

Hepatocyte Lipid Metabolism Analysis

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Dulbecco's modified Eagle's medium (DMEM), Met/Cys-free DMEM, Williams Medium E, Hepatozyme medium, fetal bovine serum (FBS), and antibiotic/antimycotic were purchased from Invitrogen Canada (Burlington, ON). Heparin, fibronectin, and fumed silica were purchased from Sigma-Aldrich (Oakville, ON). The Primaria dishes were from BD Biosciences (Mississauga, ON), and the [³⁵S]Met/Cys was obtained from MP Biochemicals (Solon, OH). Protease inhibitor cocktail and chemiluminescent substrates were purchased from Roche Diagnostics (Laval, PQ). The antibody to detect hHL (by immunoblot analysis) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), the antibodies to detect mouse apoE and apoA-I (for immunoblot and immunoprecipitation) were purchased from BioDesign International (Saco, ME, USA), and the antibodies to detect mouse calnexin and giantin were obtained from Stressgen Bioreagents (Ann Arbor, MI, USA) and Abcam (Cambridge, MA, USA), respectively. The anti-hHL antibody used for immunoprecipitation of HL was a kind gift from Dr. Ann White (University of Texas Southwestern Medical Center).

Bamji-Mirza M., Zhang W, & Yao Z. (2014). Expression of human hepatic lipase negatively impacts apolipoprotein A-I production in primary hepatocytes from Lipc-null mice. Journal of Biomedical Research, 28(3), 201-212.

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Quantification of Protein Synthesis Rates

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³⁵S-Met/Cys labeling was performed, as described previously [54 (link)]. Briefly, Rh30 or MDA-MB-231 cells, grown in 100-mm dishes to 70% confluence, were treated with CPX (0-20 μM) for 30 h or 18 h, respectively. Subsequently, the cells were briefly washed with PBS twice, and cultured in 3 ml labeling medium (DMEM, without L-Met/L-Cys, Mediatech, Herndon, VA, USA) containing 10 μM MG-132 for 10 min to deplete Met/Cys in the cells, in the presence of CPX (0-20 μM). The cells were then pulsed with 0.3 mCi ³⁵S-Met/Cys (MP Biomedicals, Solon, OH, USA) for 6 h in the presence of CPX (0-20 μM), and lysed in the RIPA buffer [50 mM Tris, pH 7.2; 150 mM NaCl; 1% sodium deoxycholate; 0.1% SDS; 1% Triton-X 100; 10 mM NaF; 1 mM Na₃VO₄; protease inhibitor cocktail (1:1000, Sigma, St. Louis, MO, USA)], followed by immunoprecipitation with antibodies to Cdc25A and GAPDH, respectively. The immunocomplexes were subjected to SDS-PAGE, transferred to a PVDF membrane, and finally autoradiographed at −80°C. NIH Image J was used for semi-quantitative analysis of the intensities of the bands.

Shen T., Shang C., Zhou H., Luo Y., Barzegar M., Odaka Y., Wu Y, & Huang S. (2017). Ciclopirox inhibits cancer cell proliferation by suppression of Cdc25A. Genes & Cancer, 8(3-4), 505-516.

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Glucose-Dependent Protein Synthesis Assay

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Following 1 hr incubation in SAB containing 2.5 mM Glc, cells were incubated for 40 min in fresh SAB containing either 2.5 mM or 12 mM Glc. Subsequently, cells were incubated for 20 min in SAB containing the respective basal or stimulatory glucose and 400 μCi of [³⁵S]-met/cys (MP Biomedical). Cells were lysed in RIPA and clarified lysates immunoprecipitated using (pro)insulin antibodies (Cell Signaling and Dako). Immunoprecipitated samples were resolved on Novex 16% Tricine gels (Life Technologies); aliquots of whole cell lysate were resolved on 4–12% NuPAGE gels (Life Technologies) for determination of total protein synthesis. Dried gels were exposed to phosphorimager screens and detected using a Typhoon Imager (GE Healthcare) and ImageQuant software (GE Healthcare).

Stephens S.B., Edwards R.J., Sadahiro M., Lin W.J., Jiang C., Salton S.R, & Newgard C.B. (2017). The prohormone VGF regulates β-cell function via insulin secretory granule biogenesis. Cell reports, 20(10), 2480-2489.

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Protein Synthesis Dynamics at Varying Temperatures

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MEF cells were incubated at 30, 37, or 39°C for 1 or 12 h. At each time point, growth medium was replaced with labeling medium (DMEM lacking cysteine and methionine [Cellgro] supplemented with 1% fetal bovine serum [FBS] and 1% penicillin-streptomycin [Pen-Strep]), and cells were returned to their temperature conditions for 15 min. Supernatants were then replaced with labeling medium containing 100 µCi/ml [³⁵S]Cys-Met (MP Biomedicals), and plates were returned to their temperature conditions for another 15 min. Cells were rinsed in PBS, and lysates were prepared with equal volumes of RIPA buffer. Twenty microliters per sample was resolved on an 8% SDS-PAGE gel, which was then fixed and dried to allow for autoradiographic signal capture of X-ray film (GE Healthcare). Gels were exposed for 7 days at −80°C, and resulting protein signal was quantified by densitometry analysis of all visible proteins in each lane using ImageJ software (NIH).

Lane W.C., Dunn M.D., Gardner C.L., Lam L.K., Watson A.M., Hartman A.L., Ryman K.D, & Klimstra W.B. (2018). The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent. mBio, 9(2), e00535-18.

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XBP1s-Induced Protein Turnover Dynamics

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HEK293^DAX-CD2 cells seeded on poly-D-lysine-coated plates were treated with Dox at 1 µg/mL to activate XBP1s for 24 h. Cells were then starved for 1 h in DMEM + 10% FBS lacking Cys and Met. Cells were metabolically labeled in pulse medium containing [³⁵S]-Cys/Met (MP Biomedicals, ~0.1 mCi/mL final concentration) for 20 min, and then incubated in pre-warmed chase media for the indicated times. Lactacystin (10 µM) was added as indicated at the beginning of the chase period. Media was collected and cells were lysed in a 1% Triton X-100 buffer. CD2 was immunopurified using M2 anti-FLAG agarose beads (Sigma; A2220) and washed with RIPA. Immunoisolates were eluted by boiling in 6X–Laemmli buffer and separated by SDS-PAGE. The gels were then dried, exposed to phosphorimager plates (GE Healthcare), and imaged with a Typhoon imager. Band intensities were quantified in ImageQuant TL.

Dewal M.B., DiChiara A.S., Antonopoulos A., Taylor R.J., Harmon C.J., Haslam S.M., Dell A, & Shoulders M.D. (2015). XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans. Chemistry & biology, 22(10), 1301-1312.

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Metabolic Labeling of Viral Proteins

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Cells were infected with viruses or transfected with plasmids for the times indicated in figure legends. Thirty minutes before labeling, growth medium was replaced with starvation medium (Cys/Met free DMEM (Cellgro) supplemented with 1% FBS, L-glut, PS). Next, cells were incubated in starvation medium complemented with 100μCi/mL [³⁵S] Cys/Met (MP Biomedicals) for 2h at 37°C. Cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer. An equal volume of sample was resolved on an 8% SDS-PAGE gel. Gels were fixed and dried and exposed to photographic film (GE Healthcare) for 7 days at −80°C for visualizing labeled proteins. Densitometry was performed as described above. As a loading control, equal volumes of lysate were resolved and stained for levels of actin using western blot.

Bhalla N., Sun C., Lam L.K., Gardner C.L., Ryman K.D, & Klimstra W.B. (2016). Host Translation Shutoff Mediated by Non-structural Protein 2 is a Critical Factor in the Antiviral State Resistance of Venezuelan Equine Encephalitis Virus. Virology, 496, 147-165.

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Transient FLuc Protein Synthesis Assay

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HEK293^HSR cells transiently transfected with FLuc were seeded on poly-D-lysine-coated plates and treated with vehicle, dox at 1 μg/mL to activate dn-cHSF1, or Shield-1 at 1 μM to activate cHSF1 for 48 h. Cells were then starved for 30 min in DMEM + 10% FBS lacking Cys and Met. Cells were metabolically labeled in pulse medium containing [³⁵S]-Cys/Met (MP Biomedical, ~0.1 mCi/mL final concentration) for 15 min prior to lysis. Cells were lysed in a 1% Triton X-100 buffer. Lysates were boiled in 1X-Laemmli buffer and separated by SDS-PAGE. The gels were then dried, exposed to phosphorimager plates (GE Healthcare), and imaged with a Typhoon imager.

Moore C.L., Dewal M.B., Nekongo E.E., Santiago S., Lu N.B., Levine S.S, & Shoulders M.D. (2015). Transportable, Chemical Genetic Methodology for the Small Molecule-Mediated Inhibition of Heat Shock Factor 1. ACS chemical biology, 11(1), 200-210.

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