For in vivo labelling cells were seeded in six-well plates in DMEM (4.5 g/L D -glucose, 41965–039, Gibco) supplemented with 10% FCS (F0804, Sigma Aldrich) with a density of 1 × 106 cells per well. The cells were starved in media without methionine and cysteine (D0422, Sigma Aldrich) for 30 min. For the labelling cells were incubated with 100 µl media containing 10% dialyzed FCS, 20 mM Hepes pH 7,4, and 25 µCi/ml [35S] methionine-label for 3.5 h at 37 °C. Afterward, cells were washed with DPBS (14190–094, Gibco) 3 times and the pellet was harvested for cell lysis. For cell lysis a buffer containing 50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2, 0.8 U/µl RNase Inhibitor (N2611, Promega), and protease inhibitor cocktail (4693132001, Roche) was used. Endogenous immunopurification was done as described above and after the separation by SDS-PAGE followed by a coomassie staining the gels were dried and analyzed by autoradiography.
D0422
Manufactured by Merck Group
D0422 is a laboratory equipment manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to facilitate sample handling and processing tasks in a research or analytical setting.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
F0804, by Merck Group (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Fla 7000, by Fujifilm (1 mentions)
Bas ms2040, by Fujifilm (1 mentions)
Ls6500, by Beckman Coulter (1 mentions)
Neg709a, by PerkinElmer (1 mentions)
7 protocols using d0422
1
Metabolic Labeling of Proteins
2
Pulse-Labeling Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pulse-labeling experiment was performed essentially as described basically performed as described (13 (link)). WT and GTPBP3 KO cells (2.0 × 106 cells) were cultured at 37°C for 15 min in methionine/cysteine-free medium [D0422 (Sigma) with 10 mM HEPES-KOH (pH 7.5), 2 mM L-glutamine, 133 μM L-cysteine and 10% FBS] containing 50 μg/ml emetine to inhibit cytoplasmic protein synthesis, followed by addition of 7.4 MBq (0.2 mCi) of [35S]-methionine/[35S]-cysteine (EXPRE35S35S Protein Labeling Mix, [35S]-, PerkinElmer), and then cultured for 1 h to specifically label mitochondrial translation products. Cell lysates (100 μg of total proteins) were resolved by Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (16.5%), and the gel was Coomassie brilliant blue (CBB)-stained and dried on a gel drier (AE-3750 RapiDry, ATTO). The radiolabeled mitochondrial protein products were visualized on an imaging plate (BAS-MS2040, Fujifilm) by a laser scanner imaging system (FLA-7000, Fujifilm).
3
Quantifying Protein Synthesis Rates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 24-well plates. Three days after the siRNA transfections, medium was removed and fresh methionine-free DMEM was added (Sigma; catalog no. D0422; supplemented with 4 mM l -glutamine). After 1.5 h, 2 μCi of [35S]methionine per 100-μl final volume was added and the cells were incubated for 3 h. WCEs were prepared as described above. Cells from two wells were combined for each reaction. A total of 50 μg of protein was transferred into a new tube, and proteins were precipitated with 100% trichloric acid for at least 10 min at 4°C. Collected pellets were then washed 3 times with 1 ml of ice-cold acetone, dried, dissolved in 100 μl of 1 N NaOH by vortexing, and finally transferred into a scintillation vial, to which 150 μl of 1 N HCl and a scintillation liquid were added. Radioactivity was measured in a scintillation counter (Beckman LS6500).
4
Radiolabeling of Newly Synthesized Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type or H19-overexpressing GH3 cells were washed with phosphate-buffered saline (PBS) and incubated in methionine and cysteine-free DMEM (Sigma, D0422) for 30 min. The 35S-labelled l -methionine and l -cysteine mix (75 μCi in a 35-mm dish) (PerkinElmer, NEG709A) was then added to the medium, and the cells were incubated for an additional 30 min. The cells were quickly washed with cold PBS and lysed in EBC buffer. Proteins were resolved by SDS-PAGE, and newly synthesized proteins were detected by autoradiography.
5
Methionine Pulse-Labeling of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated at 70% confluence. The next day, the cells were washed three times with pre-warmed PBS to remove any residual methionine and cultured for 30 min in Dulbecco’s Modified Eagle’s Medium (DMEM) without L-methionine (Sigma Aldrich, D0422). Then 100 μCi 35S labeled methionine was added to the media for a pulse labeling of 15 min. Cells were harvested after 30 min incubation with unlabeled media.
6
Extracellular GSH Analysis in BMDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In an attempt to analyze extracellular GSH levels, BMDMs or J774.1 cells were seeded in 24-well plates, both at densities of 5 × 105 cells/mL. After overnight incubation, cells were primed with LPS (100 ng/mL) for 5 h before being washed with l -methionine- and l -cystine-free DMEM (D0422, Sigma-Aldrich) supplemented with 2 mM l -glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin. Then, ATP (5 mM)-containing complete l -methionine- and l -cystine-free DMEM was added to the cells to prevent reactions between culture medium components and GSH or its metabolites. After incubation, culture supernatants and cells were harvested and analyzed with LC-MS/MS.
7
Methionine Pulse-Labeling of Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!