Flp-In 293 T-REx cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium high glucose with 10% (v/v) fetal bovine serum, 2 mM L -glutamine. Cell lines stably expressing FLAG/HA-tagged RC3H1 protein were generated by co-transfection of pFRT/TO/FLAG/HA constructs with pOG44 (Invitrogen). Cells were selected by adding 15 μg ml−1 blasticidin and 100 μg ml−1 hygromycin (Invivogen). Expression of epitope-tagged proteins was induced by addition of 1 μg ml−1 doxycyclin. The expression of FLAG/HA-tagged RC3H1 protein was assessed by western analysis using mouse anti-HA.11 monoclonal antibody (Covance). For quantitative proteomics, cells were grown in SILAC medium as described before30 (link)45 (link). Briefly, Dulbecco's modified Eagle's medium GlutaMAX lacking arginine and lysine (PAA) supplemented with 10% dialysed fetal bovine serum (Gibco) was used. Amino acids (84 mg l−1 13C615N4L -arginine plus 146 mg l−1 13C615N2L -lysine or 84 mg l-1 13C6-L -arginine plus 146 mg l−1 D4-L -lysine) or the corresponding non-labelled amino acids (Sigma) were added to obtain ‘heavy' ‘medium' or ‘light' cell culture medium, respectively. Labelled amino acids were purchased from Sigma Isotec.
13c6 15n4 l arginine
Manufactured by Merck Group
Sourced in United States
[13C6,15N4]-L-arginine is a stable isotope-labeled amino acid product manufactured by Merck Group. It is composed of L-arginine molecules with six carbon atoms and four nitrogen atoms replaced with their respective heavy isotopes, 13C and 15N. This product is intended for use in various research applications, such as metabolic studies and labeling experiments, where the tracing of arginine-derived compounds is of interest.
Automatically generated - may contain errors
Lab products found in correlation
13c6 15n2 l lysine, by Merck Group (10 mentions)
L lysine, by Merck Group (6 mentions)
L arginine, by Merck Group (5 mentions)
Methionine, by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Gentamicin, by Thermo Fisher Scientific (3 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (2 mentions)
13c6 l lysine, by Merck Group (2 mentions)
Dulbecco s phosphate buffered saline pbs, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pog44, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by InvivoGen (1 mentions)
Flp in 293 trex cells, by Thermo Fisher Scientific (1 mentions)
13c6 l arginine, by Merck Group (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Protease inhibitor cocktail tablets edta free, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Mg132, by Cayman Chemical (1 mentions)
Dialysed fbs, by Thermo Fisher Scientific (1 mentions)
14 protocols using 13c6 15n4 l arginine
1
Stable Cell Line Generation and Quantitative Proteomics
2
Stable Isotope Labeling for Protein Analysis
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Example 8
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC):
Human hepatic HuH7 cells (HuH-7), human embryonic kidney 293 cells (HEK-293) and human colorectal cancer 116 cells (HCT-116) were individually grown at 37° C. in a 5% CO2 humidified incubator. SILAC medium was prepared as follows: DMEM lacking lysine, arginine and methionine was custom prepared by AthenaES (Baltimore, Md., USA) and supplemented with 30 mg/L methionine (Sigma Aldrich; Oakville, ON, CAN), 10% (v/v) dialyzed FBS (GIBCO-Invitrogen; Burlington, ON, CAN), 1 mM sodium pyruvate (Gibco-Invitrogen), 28 μg/mL gentamicin (Gibco-Invitrogen), and [13C6,15N2]-L-lysine, [13C6,15N4]-L-arginine (heavy form of amino acids; Heavy Media) from Sigma Aldrich (Oakville, ON, CAN) at final concentrations of 42 mg/L and 146 mg/L for arginine and lysine respectively. For HCT-116, the concentration of arginine was increased to 84 mg/L. Cells were grown for at least 10 doublings in SILAC media to allow for complete incorporation of the isotopically labeled amino acids into the cells.
3
HUWE1 Gene Silencing Protocol
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Dimethyloxallyl glycine and MG132 were purchased from Cayman. CHX, Protease Inhibitor Cocktail Tablets EDTA-free, [13C6,15N2]-l -lysine, [13C6,15N4]-l -arginine, l -lysine, and l -arginine were purchased from Sigma-Aldrich. Lipofectamine RNAiMAX was purchased from Invitrogen. HUWE1 siRNA On-TARGETplus SMARTpools and negative control siRNA were purchased from Dharmacon. Benzonase nuclease lyophilized powder (CAS 9025-65-4) was purchased from Santa Cruz.
4
SILAC Labeling of Human Cell Lines
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The human hepatic HuH7 cells (HuH-7) and human embryonic kidney 293 cells (HEK-293) were originally acquired from ATCC and the human colorectal cancer 116 cells (HCT-116) were obtained from the Japanese Collection of Research Bioresources Cell Bank. HuH-7, HEK-293 and HCT-116 were individually grown at 37 °C in a 5% CO2 humidified incubator (to note, all three cell lines used in this study were found to be mycoplasma positive). Stable isotope labelling by amino acids in cell culture (SILAC) medium was prepared as follows: DMEM lacking lysine, arginine and methionine was custom prepared by AthenaES (Baltimore, MD, USA) and supplemented with 30 mg l−1 methionine (Sigma-Aldrich; Oakville, ON, Canada), 10% (v/v) dialysed FBS (GIBCO-Invitrogen; Burlington, ON,Canada), 1 mM sodium pyruvate (Gibco-Invitrogen), 28 g ml−1 gentamicin (Gibco-Invitrogen), and [13C6,15N2]-L-lysine, [13C6,15N4]-L-arginine (heavy isotopic form of amino acids; Heavy Media) from Sigma-Aldrich (Oakville, ON, Canada) at final concentrations of 42 and 146 mg l−1 for arginine and lysine, respectively. For HCT-116, the concentration of arginine was increased to 84 mg l−1. The cells were grown for at least 10 doublings in SILAC media to allow for complete incorporation of the isotopically labelled amino acids into the cells.
5
Cisplatin-Induced Apoptosis and Autophagy
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CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). 13C6-L-lysine, L-lysine, 13C615N4-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).
6
SILAC-based viral infection proteomics
7
Quantitative Comparative Cell Synchronization
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The Cdc11-TAP strain was grown in standard SD medium and the Cdc11-GFP strain in SILAC-SD medium containing 13C6,15N4-L-arginine and 13C6,15N2-L-lysine (Sigma Aldrich) as only source for arginine and lysine. Incorporation of labeled amino acids was confirmed by analyzing protein extracts from labeled cells by gel-based nano-HPLC-ESI-MS/MS analysis.
Full incorporation of the labeled amino acids was obtained by diluting a preculture grown overnight in SILAC medium into fresh SILAC medium and incubating it at 30°C for 7.5 h (S1 Fig ).
An overnight culture of each strain was diluted into 400 ml fresh medium and grown overnight at 30°C to the desired OD600nm for cell synchronization. The synchrony of the cultures was monitored by microscopy and the cells were harvested by centrifugation and immediately used for affinity purification.
Cell synchronization was performed either with alpha-factor (Sigma) (3.7 μM final concentration pre-dissolved in 0.1 M HCl; added to the cell culture at an OD600nm of 1 and incubated at 30°C for 2:40 h) or hydroxyurea (Sigma) (0.2 M final concentration; added to the cell culture at an OD600nm of 1 and incubated at 30°C for 3:30 h). Temperature-sensitive strains were arrested by incubation at 37°C for 3:25 h after the OD600nm reached 0.6.
Full incorporation of the labeled amino acids was obtained by diluting a preculture grown overnight in SILAC medium into fresh SILAC medium and incubating it at 30°C for 7.5 h (
An overnight culture of each strain was diluted into 400 ml fresh medium and grown overnight at 30°C to the desired OD600nm for cell synchronization. The synchrony of the cultures was monitored by microscopy and the cells were harvested by centrifugation and immediately used for affinity purification.
Cell synchronization was performed either with alpha-factor (Sigma) (3.7 μM final concentration pre-dissolved in 0.1 M HCl; added to the cell culture at an OD600nm of 1 and incubated at 30°C for 2:40 h) or hydroxyurea (Sigma) (0.2 M final concentration; added to the cell culture at an OD600nm of 1 and incubated at 30°C for 3:30 h). Temperature-sensitive strains were arrested by incubation at 37°C for 3:25 h after the OD600nm reached 0.6.
8
Proteomic Quantitation and SILAC Protocol
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Vancomycin, 13C6-l -lysine, l -lysine, 13C615N4-l -arginine, l -arginine, Dulbecco’s phosphate buffered saline (PBS), heat inactivate fetal bovine serum (FBS), and dialyzed FBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). DEME/F12 medium was bought from Invitrogen Inc. (Carlsbad, CA, USA). FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN, USA). The proteomic quantitation kits for acidification, desalting, and digestion, Ionic Detergent Compatibility Reagent (IDCR), DMEM/F12 medium for SILAC, Pierce bicinchoninic acid protein assay kit, and Western blotting substrate were obtained from Thermo Fisher Scientific Inc. (Hudson, NH, USA).
9
Isotopically Labeled Protein Modification
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GLP-1 7–36 amide, 13C615N2-L-lysine, 13C615N4-L-arginine, formaldehyde, 13C-D2-formaldehyde and cyanoborohydride were purchased from Sigma Aldrich (St. Louis, MO). Dialyzed heat-inactivated fetal bovine serum (FBS) was purchased from Biochrom AG (Merck, Germany). TiO2 beads were purchased from GL sciences (Tokyo, Japan).
10
Quantitative N-terminal Proteomics via SILAC
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Primary and immortalized fibroblasts were cultured in MEM SILAC growth medium supplemented with 10% dialyzed FBS and either natural 12C6-l -arginine (Sigma-Aldrich; A8094) for BJ control cells (WT or WT-hTERT cells) or 13C615N4-l -arginine (Sigma-Aldrich; 608033) for hNaa10-S37P cells (S37P or S37P-hTERT cells) at a concentration of 37.8 mg/l (i.e. 30% of the suggested concentration present in EMEM at which arginine to proline conversion was not detectable for S37P fibroblasts). Cells were cultured for at least six population doublings (14 days) to ensure complete incorporation of the labeled arginine. All free N termini were in vitro labeled by 13C2D3-acetylation and this labeling combined with differential l -arginine SILAC allows for the calculation of the degree of Nt-acetylation and for the relative quantification of N-terminal peptides between two samples (5 (link),56 (link),60 (link)). Cells (1–3 × 107) were lysed in 50 mm sodium phosphate (pH 7.5), 100 mm NaCl, 1% CHAPS, 0.5 mm EDTA and 0.5 × complete protease inhibitor cocktail (Roche) for 15 min one ice and centrifuged for 3 min at 16 000 g at 4°C, protein concentrations measured and equal amounts of control and S37P cell lysate mixed together before the lysates were subjected to N-terminal COFRADIC analysis and LC–MS/MS analysis as previously described (74 ).
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