Escherichia coli rosetta competent cells were transformed with a pNIC28 plasmid containing the BAZ2B bromodomain (residues 1868–1972), which has previously been described1 (link) and used for protein expression. 15N and 15N, 13C- uniformly labeled BAZ2B bromodomains were expressed in M9 medium9 (link) with 1.5 g of 15NH4Cl (Sigma-Aldrich) and 4.0 g of d -glucose or [U–13C6]-d -glucose (Sigma-Aldrich) per liter as the sole nitrogen and carbon sources. Colonies from freshly transformed cells were grown in 50 mL of M9 medium containing kanamycin (50 μg/mL) at 37 °C with 180 rpm shaking for 16 h. Start up cultures were diluted and inoculated in fresh M9 medium (1 L), containing kanamycin (50 μg/mL), with an initial optical densitiy (OD600) of ~0.15 and grown at 37 °C with 180 rpm shaking to an optical density (OD600) of ~0.8. At this point the temperature was decreased to 18 °C, and once the system was equilibrated protein expression was induced with 0.8 mM isopropyl-β-d -thiogalactopyranoside (IPTG) for 18 h. The cells were harvested by centrifugation (6000 rpm, 30 min, 4 °C) using a Beckman Coulter Avanti J-20 XP centrifuge.
U 13c6 d glucose
Manufactured by Merck Group
Sourced in Germany
[U–13C6]-d-glucose is a stable isotope-labeled compound that consists of a glucose molecule with all six carbon atoms labeled with the 13C isotope. This product is primarily used as a tracer in metabolic studies and research applications that require the tracking of glucose utilization and metabolism within biological systems.
Automatically generated - may contain errors
5 protocols using u 13c6 d glucose
1
Isotopic Labeling of BAZ2B Bromodomain
2
13C-Glucose Metabolite Tracing in C4-2 Cells
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C4–2 cells were plated in complete media with dialyzed FBS overnight and treated with NU7441 (1 μmol/L) or DMSO for 24 hours prior to a 2-hour pulse with [U-13C6]-D-glucose (Sigma, 389374) as described previously (24 (link)). Metabolites were extracted with ice-cold buffer (methanol: acetonitrile: MilliQ water at 50:30:20, v/v), separated on a SeQuant ZIC-pHILIC column (Merck, 150460) and identified using a Q Exactive HF mass spectrometer. Compounds were identified using an in-house library, with isotopologues corrected against naturally occurring stable carbons.
3
Metabolic Profiling of Cells Treated with DCA
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Cells were seeded in 175 cm2 culture flasks (approximately 5 × 106 cells per flask) and adhered overnight. On the following day, cells (approximately 1 × 107 cells per flask) were incubated with 75 mM of DCA for ca. 18 h and [U-13C6]glucose. Controls were treated accordingly, however with the difference that an equal volume of PBS was added and resuspended instead of the DCA solution. The [U-13C6]glucose culture medium contained unlabelled D-glucose (5 mM), [U-13C6]D-glucose (5 mM; 99.9% 13C-content, Sigma-Aldrich, Taufkirchen, Germany), glutamine (0.1 mM), glucose-free DMEM (Biochrom AG, Berlin, Germany) and 10% FCS. The cells were incubated in the incubator with 37 °C and 5% CO2. Following the defined incubation time, cells were harvested, the suspension was washed in PBS three times, and after centrifugation and removal of the supernatants, the cellular pellets were frozen at −80 °C until further use. Experiments were carried out as triplicates.
4
Isotope Labeling for Metabolic Analysis
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High glucose Dulbecco’s Modified Eagle Medium (DMEM), with Glutamax supplement and pyruvate was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Foetal bovine serum (FBS) was purchased from Invitrogen. (−)-isoproterenol hydrochloride (isoproterenol) and (S)-(−)-propranolol hydrochloride (propranolol) were purchased from Sigma Aldrich (St. Louis, MO, USA). For the isotope labelling studies, U-13C6-d -Glucose was purchased from Sigma Aldrich and U-13C5-l -Glutamine was purchased from NovaChem. All other solvents and reagents were of LC-MS analytical grade (Merck, Kenilworth, NJ, USA).
5
Metabolic Profiling of Targeted Radiotherapy
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Cells were seeded in 175 cm2 culture flasks (approximately 5 × 106 cells per flask) and allowed to adhere overnight. The next day, cells (approximately 1 × 107 cells per flask) were incubated with lethal activity concentrations of 1.48 MBq/ml of 213Bi-anti-EGFR-MAb in a total volume of 10 ml culture medium for 3 h at 37 °C and 5% CO2. Controls were treated accordingly, however, an equal volume of PBS was added instead of 213Bi-anti-EGFR-MAb solution. Subsequently, culture medium was aspirated and the cells were washed once with 20 ml of PBS. Following addition of 30 ml per flask of [U-13C6]glucose containing culture medium (glucose-free DMEM [Biochrom, Berlin, Germany] with added unlabelled d -glucose [5 mM], [U-13C6]d -glucose [5 mM] [99.9% 13C-content, Sigma-Aldrich, Taufkirchen, Germany], glutamine [0.1 mM] and 10% FCS), the cells were incubated for another 18 h in the incubator (37 °C, 5% CO2). Finally, cells were harvested, washed in PBS three times and the cellular pellets (after removal of the supernatants) were frozen at − 80 °C until further use. Analysis of the cells was done after these 18 h of incubation with [U-13C6]glucose.
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