Human ovarian carcinoma cell line OVCAR-3 (ATCC, HTB161) was obtained from ATCC (Rockville, MD). The cell line was tested for mycoplasma contamination by ATCC before being used in experiments. A total of 32 dishes (15 cm × 15 cm) of OVCAR-3 cells were cultured in RPMI 1640 medium with 10% FBS to 50% confluence, and half of the dishes were treated with 1 µM tunicamycin for 48 h to inhibit N-glycosylation. In order to analyze the glycoprotein changes in tunicamycin-treated cells, the OVCAR-3 cells were also treated with 1 µM tunicamycin in triplicate and collected at six different time points (0, 6, 12, 24, 48 and 72 h). Separate dishes of OVCAR-3 cells were cultured in SILAC RPMI 1640 medium containing heavy isotope-labeled 13C6-l -lysine and 13C615N4-l -arginine (Cambridge Isotope Laboratories, Andover, MA) supplemented with 10% dialyzed FBS (diFBS) (Invitrogen, Carlsbad, CA) to generate heavy SILAC-labeled (K6, R10) OVCAR-3 cells. The cells were cultured for approximately ten doublings in the SILAC medium to ensure complete labeling31 . After removing the medium, the cells were washed five times with phosphate buffered saline (PBS, pH 7.4) buffer and then lysed directly with 8 M urea/1 M NH4HCO3 solution32 . Lysates were briefly sonicated until the solutions were clear. Protein concentrations were determined by BCA protein assay reagent (Thermo Scientific, Fair Lawn, NJ).
13c615n4 l arginine
Manufactured by Cambridge Isotopes
Sourced in United States
The 13C615N4 L-arginine is a stable isotope-labeled amino acid product. It contains carbon-13 and nitrogen-15 isotopes, which can be used as tracers in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
L lysine 13c615n2, by Cambridge Isotopes (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
13c6 l arginine, by Cambridge Isotopes (4 mentions)
13c6 l lysine, by Cambridge Isotopes (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
L arginine, by Cambridge Isotopes (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
L lysine, by Thermo Fisher Scientific (2 mentions)
L arginine, by Thermo Fisher Scientific (2 mentions)
Ovcar3, by American Type Culture Collection (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ovcar3, by Thermo Fisher Scientific (1 mentions)
Silac, by Cambridge Isotopes (1 mentions)
13c615n2 l lysine lys8, by Cambridge Isotopes (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L proline, by Merck Group (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Hela s3 cells, by Thermo Fisher Scientific (1 mentions)
18 protocols using 13c615n4 l arginine
1
OVCAR-3 Glycoproteomic Analysis
2
Primed vs Naive hESC Proteomics
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To perform quantitative mass spectrometry based whole-proteome comparison of primed and naive pluripotent states, WIBR2 and WIBR3 primed hESCs were cultured in SILAC heavy medium whereas chemically reset naïve hESCs were cultured in 4i/L/A naïve medium (SILAC light). For the SILAC heavy condition, primed hESCs were grown for three passages in DMEM/F12 with corresponding complete supplements but deficient in both L-lysine and L-arginine and supplemented with heavy 13C615N4 L-arginine and 13C615N2 L-lysine (Cambridge Isotope Laboratories). The medium was supplemented with 10% dialyzed FBS for SILAC (Thermo Fisher Scientific), and all other gradients followed the normal condition for primed hESC culture.
3
Triplex SILAC Labeling of Bladder Smooth Muscle Cells
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Primary human bladder smooth muscle cells (pBSMCs) were cultured in smooth muscle cell medium (SMCM, Sciencell Research Laboratories, Carlsbad, CA) at 37°C in a humidified incubator with 5% CO2. For triplex SILAC labeling, pBSMCs were grown in arginine- and lysine-depleted SMCM (Sciencell Research Laboratories) supplemented with 2% (v/v) dialyzed fetal bovine serum (Invitrogen, Grand Island, NY) and L-arginine (Arg0) and L-lysine (Lys0), 13C6-L-arginine (Arg6) and 4,4,5,5-D4-L-lysine (Lys4), or 13C615N4-L-arginine (Arg10) and 13C615N2-L-lysine (Lys8) (Cambridge Isotope Laboratories, Andover, MA). After at least 6 population doublings, pBSMCs cultured in “light”, “medium”, and “heavy” SILAC media were serum starved overnight and treated with 1 nM PDGF-BB for 0, 4, and 24 h, respectively.
4
Differentiation of iPSCs into Atrial and Ventricular Cardiomyocytes
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Human iPSC-lines from two healthy donors were used in this study. Human iPSC lines UMGi001-A clone 1 (iWT.D2.1) and UMGi014-A clone 2 (isWT1.Bld2) were described previously16 (link). These cell lines had been generated from dermal fibroblasts and peripheral blood mononuclear cells, respectively, using the STEMCCA lentivirus system or the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit. Each of the two iPSC lines were differentiated into atrial and ventricular iPSC-CMs via modulation of WNT signaling and retinoic acid modulation and subsequent metabolic selection, as previously described16 (link). Differentiated cultures were labeled with stable isotope-labeled arginine and lysine for 45 days in RPMI1640 (ThermoFisher), 2% B27 (ThermoFisher), 10 μl/ml Glutamax (ThermoFisher), 25 mM HEPES (ThermoFisher), 1.74 mM l -proline (Sigma-Aldrich), 0.219 mM 13C6,15N2-l -lysine and 0.575 mM 13C6,15N4-l -arginine (Cambridge Isotopes). SILAC-labeled cells were pelleted at day 65–69 of differentiation. Differentiation of iPSCs into atrial and ventricular CMs was performed in three separated replicates each, respectively. Proteins were isolated, protein concentrations determined, and equal amounts of protein mixed to generate the quantification standard to obtain equal representation of both atrial and ventricular proteins in the standard16 (link),21 (link),22 (link).
5
SILAC Labeling of SKOV3 Ovarian Cells
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SKOV3 ovarian carcinoma cells (ATCC HTB-77) originating from the same stock were split into two, one set was cultured in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco) and the other in RPMI 1640 media for SILAC (Cambridge Isotope Laboratories) that was supplemented with 10% dialyzed FBS (Cambridge Isotope Laboratories), 120 mg/L 13C615N4l -arginine (Cambridge Isotope Laboratories) and 40 mg/L 13C6l -lysine (Cambridge Isotope Laboratories). Both SKOV3 cell populations were maintained at the same passage and cultured under the same conditions (37 °C, 5% CO2). Incorporation of the isotopically heavy arginine and lysine was allowed to exceed 98% as determined by LC–MS/MS analysis of trypsin digested heavy SKOV3 lysate.
6
Constructing Yeast Strain TYSC110 for SILAC
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Saccharomyces cerevisiae haploid strain TYSC110 (MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 lys2Δ0 arg4Δ::kanMX4) was constructed by crossing EUROSCARF strains BY4742 (MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0) and arg4Δ (MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ YHR018c::kanMX4) followed by diploid isolation, sporulation, tetrad dissection and selection of a lysine and arginine auxotroph.
Yeast cells were grown at 30°C in „light“ synthetic minimal (SD) media (0.67% yeast nitrogen base without amino acids, 2% glucose) supplemented with 20 mg/l L-histidine, 60 mg/l L-leucine, 20 mg/l L-methionine, 20 mg/l uracil, 30 mg/l L-lysine and 20 mg/l L-arginine [10] (link) to mid log phase (OD600 of 1.8).
For lysine and arginine double SILAC labeling [11] (link), L-lysine and L-arginine in the media were substituted with isotopically heavy 30 mg/l 13C615N2 L-lysine and 20 mg/l 13C615N4 L-arginine (Cambridge Isotope Laboratories), respectively. Cells were grown in “heavy” medium at 30°C for 16 h (approximately 10 generations) to mid log phase (OD600 of 1.4).
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Saccharomyces cerevisiae haploid strain TYSC110 (MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 lys2Δ0 arg4Δ::kanMX4) was constructed by crossing EUROSCARF strains BY4742 (MATα his3Δ1 leu2Δ0 ura3Δ0 lys2Δ0) and arg4Δ (MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ YHR018c::kanMX4) followed by diploid isolation, sporulation, tetrad dissection and selection of a lysine and arginine auxotroph.
Yeast cells were grown at 30°C in „light“ synthetic minimal (SD) media (0.67% yeast nitrogen base without amino acids, 2% glucose) supplemented with 20 mg/l L-histidine, 60 mg/l L-leucine, 20 mg/l L-methionine, 20 mg/l uracil, 30 mg/l L-lysine and 20 mg/l L-arginine [10] (link) to mid log phase (OD600 of 1.8).
For lysine and arginine double SILAC labeling [11] (link), L-lysine and L-arginine in the media were substituted with isotopically heavy 30 mg/l 13C615N2 L-lysine and 20 mg/l 13C615N4 L-arginine (Cambridge Isotope Laboratories), respectively. Cells were grown in “heavy” medium at 30°C for 16 h (approximately 10 generations) to mid log phase (OD600 of 1.4).
7
Proteomic Profiling of HIV Infection in T Cells
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A population of 500 × 106 SupT1 cells (lymphoblastic T cell line) was cultured in RPMI 1640 medium with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen) (Fig. 1 ). Isotope-labeled amino acids (13C6-L-lysine, 13C615N4-L-arginine, Cambridge Isotope Laboratories (CIL), Andover, MA) were included in the heavy (H) SILAC medium at 100 mg/l, while normal Arginine and Lysine were used in the light (L) SILAC medium. Heavy or light SILAC labeling was achieved by culturing the cells in the two media (H and L) for a minimum of 2 weeks to allow for at least 5 cell divisions. H-labeled cells were Mock infected, while the L-labeled cells were infected with an HIVeGFP/VSV-G virus at 3 μg/106 cells. Infection (both Mock and with an HIVeGFP vector) was carried out by spinoculation for 30 min at 1500 g in presence of 5 μg/ml polybrene. As previously described18 (link), this allowed reaching a quasi-universal infection. Cells were then washed and further incubated. The HIVeGFP viral vector used expresses GFP instead of the viral protein env. At multiple time points post-infection, cells were collected and processed for analysis of HIV life cycle progression and normalized by the 24 h time point as in18 (link), as well as for transcriptome, proteome and phosphoproteome as detailed below (Supplementary File S10 ).
8
SILAC Labeling of HeLa S3 Cells
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HeLa S3 cells were purchased from the American Type Culture Collection, as detailed in Key Resources Table, and cultured at 37°C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). For SILAC, HeLa S3 cells were grown at 37°C in a humidified atmosphere with 5% CO2 in DMEM (without lysine and arginine; Life Technologies) containing 10% FBS (Life Technologies) and penicillin-streptomycin and supplemented with 13C615N4-l -arginine (22 mg/liter; Cambridge Isotope Laboratories) and 13C615N4-l -lysine (50 mg/liter; Cambridge Isotope Laboratories) (heavy medium) or the corresponding nonlabeled amino acids (Peptide International) (light medium).
9
SILAC-Based Proteomic Analysis of GPR124
10
SILAC Labeling of HeLa S3 Cells
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HeLa S3 cells were grown in suspension at 37°C in a humidified atmosphere with 5% CO2 in DMEM medium (–Arg, –Lys; Life Technologies) containing 10% dialyzed fetal bovine serum (Life Technologies), penicillin–streptomycin, and supplemented with 22 mg/L 13C615N4-L-arginine (Cambridge Isotope Laboratories, Tewksbury, MA) and 50 mg/L 13C615N2-L-lysine (Cambridge Isotope) or the corresponding non-labeled amino acids (Peptide International, Louisville, KY). Harvested cell pellets were washed with ice cold phosphate buffered saline (PBS) and frozen in liquid N2. The cell powder grinded with a Ball Mill (Retch MM301) was stored at −80 °C until use.
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