35s cysteine

Manufactured by American Radiolabeled Chemicals

[35S]cysteine is a radioactive amino acid that contains the radioactive isotope sulfur-35. It is commonly used in various scientific and research applications that require the incorporation of a radioactive label into proteins or other biomolecules.

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Lab products found in correlation

35s methionine, by American Radiolabeled Chemicals (3 mentions) Typhoon fla 9500, by GE Healthcare (2 mentions) Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Typhoon trio, by GE Healthcare (1 mentions) Fla 5000, by Fujifilm (1 mentions)

4 protocols using 35s cysteine

Monitoring Sulfur Transfer in ISCU2/FXN Complexes

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Reaction
mixtures (30 μL) for monitoring sulfur transfer included 3 μM
SD, 9–120 μM ISCU2, buffer A, and either 0 or 9 μM
FXN. A similar experiment was performed in which FRDA FXN variants
N146K, Q148R, I154F, W155R, and R165C (each at 9 μM), which
were purified as previously described,^{24 (link),25 (link)} were substituted
for native FXN. “Hot” l-cysteine (100 μM)
was prepared by adding 50 Ci/mmol [³⁵S]cysteine (American
Radiolabeled Chemicals Inc.) to a 1 mM “cold” cysteine
stock solution. The hot l-cysteine was added to the samples
and reacted for 2 min at 37 °C, and the reactions were terminated
by centrifugation through a Micro Bio-Spin P-6 gel filtration column
(Bio-Rad). The spin column flow-through was combined with nonreducing
SDS–PAGE sample loading buffer and then loaded on a nonreducing
14% SDS–PAGE gel. The gel was dried on chromatography paper
in a gel-drying oven at 60 °C under vacuum before a 12 h exposure
to a phosphor screen. Incorporation of the ³⁵S label was
visualized using a Phosphorimager (Typhoon Trio, GE Healthcare).

Bridwell-Rabb J., Fox N.G., Tsai C.L., Winn A.M, & Barondeau D.P. (2014). Human Frataxin Activates Fe–S Cluster Biosynthesis by Facilitating Sulfur Transfer Chemistry. Biochemistry, 53(30), 4904-4913.

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Pulse-Chase Labeling and Fractionation of CHO-K1 Cells

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CHO-K1 cells growing in 35-mm dishes were washed twice with PBS, incubated in cysteine- and methionine-free DMEM for 30 min, and then pulse labeled for 1 h with 10 mCi/ml [³⁵S]methionine plus [³⁵S]cysteine (American Radiolabeled Chemicals). To chase the ³⁵S-labeled proteins, cells were washed with PBS and further incubated with complete Ham's F12 medium containing 10% FBS for 12 h. Cells were transfected with empty vector, BAK-P26, or BAK-P26–L78A, and were further incubated for 12 h. ³⁵S-labeled cells were harvested and incubated in homogenizing buffer containing 25 µg/ml digitonin for 5 min at room temperature as described previously (Natsuyama et al., 2013 (link); or origin). After centrifugation at 100,000 g for 30 min at 4°C, cytosolic and organellar fractions were subjected to immunoprecipitation with anticatalase antibody as described previously (Tsukamoto et al., 1990 (link)). ³⁵S-labeled proteins were separated on SDS-PAGE (9% gel) and detected with an Autoimaging analyzer (FLA-5000; Fujifilm).

Hosoi K.I., Miyata N., Mukai S., Furuki S., Okumoto K., Cheng E.H, & Fujiki Y. (2017). The VDAC2–BAK axis regulates peroxisomal membrane permeability. The Journal of Cell Biology, 216(3), 709-722.

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Metabolic Labeling and Fractionation of Cellular Proteins

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Fao cells growing in DMEM supplemented with 10% FBS in 6-well plate were washed twice with PBS, incubated in cysteine- and methionine-free DMEM (Gibco) supplemented with 10% FBS that had been dialyzed for 1 hr in excess PBS with a Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). Cells were then pulse-labeled for 1 hr by adding 100 μCi/ml ³⁵S-methionine plus ³⁵S-cysteine (American Radiolabeled Chemicals). To chase the ³⁵S-labeled proteins, cells were washed twice and further incubated for 1 hr with DMEM supplemented with 10% FBS and 10 mM methionine. ³⁵S-labeled cells were harvested and incubated for 5 min in buffer H containing 50 μg/ml digitonin at room temperature as described (Natsuyama et al., 2013 (link)). After centrifugation at 20,000 g for 30 min at 4°C, cytosolic and organelle fractions were subjected to immunoprecipitation with antibodies to catalase and DHAPAT as described (Tsukamoto et al., 1990 (link)). ³⁵S-labeled proteins were separated by SDS-PAGE and detected with an Autoimaging analyzer (Typhoon FLA-9500; GE Healthcare).

Okumoto K., El Shermely M., Natsui M., Kosako H., Natsuyama R., Marutani T, & Fujiki Y. (2020). The peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation. eLife, 9, e55896.

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Subcellular Fractionation and Protein Labeling

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Fao cells growing in DMEM supplemented with 10 % FBS in 6-well plate were washed twice with PBS, incubated in cysteine-and methionine-free DMEM (Gibco) supplemented with 10%
FBS that had been dialyzed for 1 h in excess PBS with Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). Cells were then pulse-labeled for 1 h by adding 100 Ci/ml 35 S-methionine plus 35 S-cysteine (American Radiolabeled Chemicals). To chase the 35 S-labeled proteins, cells were washed twice and further incubated for 1 h with DMEM supplemented with 10 % FBS and 10 mM methionine. 35 S-labeled cells were harvested and incubated for 5 min in buffer H containing 50 g/ml digitonin at room temperature as described (Natsuyama et al., 2013) . After centrifugation at 20,000 g for 30 min at 4°C, cytosolic and organelle fractions were subjected to immunoprecipitation with antibodies to catalase and DHAPAT as described (Tsukamoto et al., 1990) . 35 S-labeled proteins were separated by SDS-PAGE and detected with an Autoimaging analyzer (Typhoon FLA-9500; GE Healthcare).

Okumoto K., Shermely M.E., Natsui M., Kosako H., Natsuyama R., Marutani T., & Fujiki Y. (2020). Peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation.

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