35s cys

Manufactured by PerkinElmer

Sourced in United States

[35S]Cys is a radioisotope-labeled amino acid that can be used for various biological and biochemical applications, such as protein labeling and detection. It contains the radioactive isotope sulfur-35 (35S) and the cysteine amino acid. The core function of [35S]Cys is to serve as a tracer or label for the detection and analysis of proteins and other biomolecules.

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Close spelling variants of this product

[35S]Met and [35S]Cys protein labeling mix EasyTag Express35S Protein Labeling Mix [35S]-Cys/Met Promix cell labeling mix ([35S]Met plus [35S]Cys), [35S]Met/Cys EXPRE35S35S protein labelling mix 35S-Met/Cys 35S-Met/Cys radiolabel [35S]Met/Cys pro-mix

5 protocols using 35s cys

Human Microsomes Compound Metabolism Assay

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Pooled human microsomes (n = 50, mixed gender) were purchased from Sekisui XenoTech, LLC (Kansas City, KS, USA). NADPH-generating system was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Troglitazone, clozapine, indinavir and acetonitrile (MeCN) were purchased from Wako Pure Chemicals (Osaka, Japan). Nefazodone hydrochloride, buspirone hydrochloride, ciprofloxacin, diclofenac sodium, ketoconazole and carbamazepine were purchased from Sigma-Aldrich (St Louis, MO, USA). Quetiapine fumarate was purchased from Toronto Research Chemicals (North York, Canada). Rimonabant was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). [ 35 S]Cys, [ 14 C]KCN, Ultima FLO-M, and Deepwell LumaPlate-96 were purchased from PerkinElmer Life and Analytical Science (Boston, MA, USA). XBridge BEH C18 Column, 130Å, 3.5 µm, 4.6 mm × 150 mm and Sep-Pak tC18 96-well µElution Plates were purchased from Waters Corporation (Milford, MA, USA). Formic acid (FA) and dimethyl sulfide (DMSO) were purchased from Nacalai Tesque (Kyoto, Japan).

Kakutani N., Nanayama T, & Nomura Y. (2019). Novel risk assessment of reactive metabolites from discovery to clinical stage. The Journal of toxicological sciences, 44(3).

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Protein Purification and Antibody Production

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The E. coli strains (Table S1), plasmids (Table S2), and PCR primers (Table S3) used in this study are listed. SecA^{40 (link)} and SecB^{41 (link)} were purified as described. Antibodies against MPIase^{7 (link)}, SecB^{42 (link)}, SecD^{27 (link)}, SecG^{43 (link)} and OmpA^{44 (link)} were raised in rabbits using the respective purified factors, while those against CdsA (Glu75~Ser92), M13 procoat (Ala24~Tyr44), LolC (Lys239~Glu255)^{28 (link)}, and YidC (Lys382~Gln402) were raised against synthetic peptides corresponding to the specified regions. All the antibodies were obtained through the commercially available custom services. [Glycerol-¹⁴C(U)]-L-α-dipalmitoyl-phosphatidic acid (3.7~7.4 GBq/mmol), [¹⁴C(U)] palmitic acid (>18.5 GBq/mmol), and [³⁵S] EXPRESS Protein Labeling Mix, a mixture containing both [³⁵S] Met and [³⁵S] Cys (~37 TBq/mmol), were obtained from Perkin Elmer, Inc. CDP-DAG, PA, PG and PE were from Avanti Polar Lipids, Inc. CL, GlcNAc-P, UDP-GlcNAc and L-arabinose were obtained from Sigma-Aldrich. CTP was from Roche Diagnostics. IPTG was from Wako Pure Chemical Industries, Ltd. CDP-GlcNAc (Supplementary Fig. 18) and the authentic reference of compound I (Supplementary Fig. 19) were chemically synthesized as described under Supplementary Methods. Protein A Sepharose was from GE Healthcare Life Sciences. TLC plates were from Merck.

Sawasato K., Sato R., Nishikawa H., Iimura N., Kamemoto Y., Fujikawa K., Yamaguchi T., Kuruma Y., Tamura Y., Endo T., Ueda T., Shimamoto K, & Nishiyama K.I. (2019). CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo. Scientific Reports, 9, 1372.

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Metabolic Labeling of Cells

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PF and purified MF cells (4 × 10⁷ each) were collected and resuspended in 4 ml Cunningham’s medium prepared without Met and Cys and supplemented with 2 mM L-Gln and 10% dialyzed FBS. Each cell sample was aliquoted into four 1.5 ml Eppendorf tubes and incubated at room temperature for 30 min. Cells were spun down and resuspended in 1 ml of the minus Met/Cys, dialyzed serum medium. To each tube, 5 μl (~50 μCi) of protein labeling mix containing both [³⁵S]Met and [³⁵S]Cys (PerkinElmer) were added and samples were incubated at 28°C for the indicated time. Cells were washed twice with PBSG (PBS containing 0.7 g l⁻¹ glucose), lysed with 200 μl of SDS-PAGE loading buffer, and 20 μl were separated on 10% SDS-PAGE. The gel was stained with Coomassie, destained, dried, and exposed for analysis with Typhoon FLA 7000 PhosphorImager (GE Healthcare Life Sciences).

Christiano R., Kolev N.G., Shi H., Ullu E., Walther T.C, & Tschudi C. (2017). The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion. Molecular microbiology, 106(1), 74-92.

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Pulse Labeling of Protein Synthesis

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MIN6 cells or mouse islets were cultured in 12-well plates in the presence or absence of palmitate or cycloheximide for 1 h. For the pulse label, the cells or islets were washed and incubated with culture media lacking Met and Cys, supplemented with 125 μCi of a mixture of ³⁵S-Met and ³⁵S-Cys (PerkinElmer) for the final 15 min of the incubation. Cellular lysates were loaded on a 10% SDS-polyacrylamide gel and subjected to electrophoresis. The gel was dried and exposed to X-ray film overnight and quantitated using ImageJ Software (National Institutes of Health).

Hatanaka M., Maier B., Sims E.K., Templin A.T., Kulkarni R.N., Evans-Molina C, & Mirmira R.G. (2014). Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway. Diabetes, 63(10), 3404-3415.

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Melanoma Cell Cysteine Uptake

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Rates of Cys and cystine uptake by the melanoma cells were calculated after administering i.v. 2.0 μCi of [³⁵S]Cys or 10.0 μCi of [³⁵S]cystine (PerkinElmer, Waltham, MA, USA).

Obrador E., Salvador-Palmer R., López-Blanch R., Oriol-Caballo M., Moreno-Murciano P, & Estrela J.M. (2022). N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice. Cancers, 14(15), 3614.

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