For generation of 15N-labeled REV-ERBβ LBD, protein was expressed in BL21(DE3) E. coli cells using M9 minimal media supplemented with 15NH4Cl (Cambridge Isotope Laboratories) induced at an optical density at 600 nm (OD600) of 0.6 with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) for 16 hours at 18°C. For generation of 2H-,15N-,13C-labeled REV-ERBβ LBD (~70% deuteration) for peak assignment experiments, cultures were first grown at 37°C in 1 liter of LB media until an OD600 of 0.6 before the cells were pelleted and resuspended in 0.5 liters of M9 minimal media prepared in D2O supplemented with 13C-d -glucose and 15NH4Cl (Cambridge Isotope Laboratories). After 1-hour recovery at 37°C, expression was induced with 0.5 mM IPTG for 16 hour at 37°C before harvesting by centrifugation. The purifications were performed as described for unlabeled protein.
15nh4cl
Manufactured by Cambridge Isotopes
Sourced in United States
15NH4Cl is a stable isotope compound containing the nitrogen-15 isotope. It is used as a tracer in various analytical and research applications.
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Lab products found in correlation
13c glucose, by Cambridge Isotopes (13 mentions)
13c labeled glucose, by Cambridge Isotopes (7 mentions)
13c d glucose, by Cambridge Isotopes (3 mentions)
Dmso d6, by Cambridge Isotopes (3 mentions)
Superdex 75, by GE Healthcare (3 mentions)
13c6 d glucose, by Cambridge Isotopes (2 mentions)
Bme vitamin mix, by Merck Group (2 mentions)
Chelex 100, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Luria broth, by Avantor (2 mentions)
U 13c glucose, by Cambridge Isotopes (2 mentions)
M9 minimal medium, by Cambridge Isotopes (2 mentions)
U 13c6 glucose, by Cambridge Isotopes (2 mentions)
Pet 50b derivative plasmid, by GenScript (2 mentions)
Kta pure 25 system, by GE Healthcare (2 mentions)
Lm10 microfluidizer, by Microfluidics (2 mentions)
Probond, by Thermo Fisher Scientific (2 mentions)
Bioflo 415, by Eppendorf (2 mentions)
Vinyl chloride, by Merck Group (2 mentions)
Ethene, by Merck Group (2 mentions)
94 protocols using 15nh4cl
1
Preparation of Isotopically Labeled REV-ERBβ LBD
2
Anammox-CdS Biohybrid System Tracer Study
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The 15N stable isotope tracer experiment was used to explore the pathway of NH4+-N conversion in the anammox-CdS biohybrid system. After anammox biomass was combined with the CdS nanoparticles, the supernatant was poured out, and the labeled 15NH4Cl of 25 mg-N/L (Cambridge Isotope Laboratories, Trading Company, USA), 0.1 g cystine were added to the vial along with 100 mL medium incubated for 9 h (sparged with helium to ensure anaerobic conditions in the vial), and with gas sample analysis every 3 h. The concentration of 28N2, 29N2, 30N2, 14NO, 15NO, 28N2O, 29N2O and 30N2O were measured by a gas chromatography-mass spectrometer (GCMS-QP, SHIMADZU, Japan).
To exploit the real intermediate product during photocatalytic NH4+-N oxidation process, the anammox-CdS biohybrid system was incubated with 15NH4Cl of 25 mg-N/L (Cambridge Isotope Laboratories, Trading Company, USA) and 14NH2OH of 12.5 mg-N/L for 9 h, and extracted the headspace gas every 3 h to detect the content of 30N2, 29N2 and 28N2 produced by the gas chromatography-mass spectrometer (GCMS-QP, SHIMADZU, Japan).
To exploit the real intermediate product during photocatalytic NH4+-N oxidation process, the anammox-CdS biohybrid system was incubated with 15NH4Cl of 25 mg-N/L (Cambridge Isotope Laboratories, Trading Company, USA) and 14NH2OH of 12.5 mg-N/L for 9 h, and extracted the headspace gas every 3 h to detect the content of 30N2, 29N2 and 28N2 produced by the gas chromatography-mass spectrometer (GCMS-QP, SHIMADZU, Japan).
3
Protein Expression in E. coli BL21(DE3) Rosetta
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E. coli BL21(DE3) Rosetta cells (SGC Frankfurt) were transformed with the respective E. coli expression plasmids for protein production. Cells were grown at 37 °C in 2xYT medium supplemented with 100 µM zinc acetate (Carl Roth) to an optical density (OD) of ~0.8. Protein expression was induced with 1 mM IPTG (Carl Roth) for 16–18 h at 18 °C. For labelled expression, cells were grown in LB medium at 37 °C to an OD of ~1, harvested by centrifugation and resuspended in M9 minimal medium supplemented with 100 µM zinc acetate and the appropriate isotopic labelling reagents (1 g/l 15NH4Cl (Cambridge Isotope Laboratories Inc.) and 4 g/l glucose (Carl Roth) for 15N-labelling; 1 g/l 15NH4Cl and 2 g/l 13C-glucose (Cambridge Isotope Laboratories Inc.) for 15N/13C-labelling) to an OD of ~0.3. Cells were then grown at 37 °C to an OD of ~0.8 and protein expression was carried out as described above.
4
Antibody and Reagent Characterization for Protein Signaling
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Mouse anti-β-arrestin-1 polyclonal antibody was from Abmart. Rabbit anti-β-arrestin-1 (A1CT) antibody was a gift from Dr. Robert J. Lefkowitz. Rabbit anti-PPARα polyclonal antibody was purchased from Abcam. Rabbit anti-PPARγ polyclonal antibody was from Santa Cruz Biotechnology. Thrombin protease was obtained from GE Healthcare. 15NH4Cl was from Cambridge Isotope Laboratories. Inc. Rosiglitazone, GW7647, and n-Dodecyl β-D-maltoside (DDM) were from Sigma.
5
Uniform Isotopic Labeling and Purification of IDR1-NTD-IDR2 Protein
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Uniformly 13C, 15N-labelled IDR1-NTD-IDR2 protein was expressed in E. coli strain BL21 (DE3). The culture was grown in 1 L LB medium at 37 °C until OD600 reached 0.8, then transferred in 250 mL of labelled minimal medium (4x) containing 0.25 g/L 15NH4Cl (Cambridge Isotope Laboratories), 0.75 g/L [U]13C6-d -glucose (Eurisotop). After 1 h of metabolite clearance, the culture was induced with 0.2 mM isopropyl-beta-thiogalactopyranoside (IPTG) at 18 °C for 16/18 h.
The cell pellet was resuspended in 25 mM 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), 1.0 M sodium chloride, 5% glycerol, DNAse, RNAse and 500 µL of 100 × stock of protease inhibitor cocktail (SIGMA) at pH 8.
Cells were disrupted by sonication. The supernatant was cleared by centrifugation (50′, 30,000×g, 4 °C), then the cleared supernatant was dialyzed overnight at 4 °C into 25 mM TRIS pH 7.2 (binding buffer).
The protein was purified with ion-exchange chromatography using an HiTrap SP FF 5 mL column and a 70% gradient of 25 mM TRIS, 1 M NaCl pH 7.2. Fractions containing pure protein were pooled and concentrated using 15 mL and 0.5 mL Centricon centrifugal concentrators (MW cutoff 10 kDa).
Final NMR samples were 280 µM IDR1-NTD-IDR2, 25 mM TRIS pH 6.5, 450 mM sodium chloride, 0.02% NaN3, 5% (v/v) D2O in water.
The cell pellet was resuspended in 25 mM 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), 1.0 M sodium chloride, 5% glycerol, DNAse, RNAse and 500 µL of 100 × stock of protease inhibitor cocktail (SIGMA) at pH 8.
Cells were disrupted by sonication. The supernatant was cleared by centrifugation (50′, 30,000×g, 4 °C), then the cleared supernatant was dialyzed overnight at 4 °C into 25 mM TRIS pH 7.2 (binding buffer).
The protein was purified with ion-exchange chromatography using an HiTrap SP FF 5 mL column and a 70% gradient of 25 mM TRIS, 1 M NaCl pH 7.2. Fractions containing pure protein were pooled and concentrated using 15 mL and 0.5 mL Centricon centrifugal concentrators (MW cutoff 10 kDa).
Final NMR samples were 280 µM IDR1-NTD-IDR2, 25 mM TRIS pH 6.5, 450 mM sodium chloride, 0.02% NaN3, 5% (v/v) D2O in water.
6
NMR Spectroscopy of Isotopically Labeled Proteins
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Ampicillin, SDS, acetonitrile (high performance liquid chromatography (HPLC) grade), and reagents to make lysogeny broth (LB) medium were purchased from Fisher Scientific (Ottawa, ON). DPC and LPPG were purchased from Anatrace (Maumee, OH) and Avanti Polar lipids (Alabaster, AL), respectively. 15NH4Cl, 13C6 -d-glucose, SDS-d25, and DPC-d38 were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Deuterium oxide (D2O; 99.8 atom % D) and D2O containing 1% (w/w) sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) were obtained from C/D/N Isotopes (Pointe-Claire, QC). All other reagents were purchased from Sigma-Aldrich Canada (Oakville, ON).
7
Labeled Protein Production in E. coli
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Similar to our previously published method of labeled protein production24 (link), E. coli bacteria were grown in M9 minimal media with 99% 15NH4Cl (Cambridge Isotope Laboratories) as the sole nitrogen source. For this growth, cells were grown at 37 °C in four 25 ml cultures overnight at 180 rpm. The following day, 0.5 L flasks were started with 25 mls of the overnight culture, and were incubated at 37 °C and 200 rpm until an OD600 of 1.0 then the temperature was reduced to 22 °C. After letting the cells equilibrate to the new temperature for 1 h protein expression was induced by the addition of 500 µM IPTG and cells were harvested after 16 h of additional incubation. Protein expression and purification was then done the same as for the 19F labeled protein.
8
Stable Isotope Labeling and NMR Analysis of DDX3X
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15N-labeled D2 domain of DDX3X WT and L556S was produced in M9 minimal medium supplemented with 1g/L 15NH4Cl (Cambridge Isotope Laboratories). The expression and purification protocols were performed as described for the unlabeled protein. The gel-filtration final buffer contained 10 mM Hepes, 500 mM ammonium sulfate and 1 mM DTT. Labeled protein was concentrated and 10% D2O was added. NMR spectra were recorded at 12°C on an Agilent Inova 500-MHz spectrometer. The spectra were processed with NMRPipe/NMRDraw and analyzed with NMRView (Johnson, 2004 (link)).
9
Expression and Purification of Hdm2-ABD
10
Isotopic Labeling of SARS-CoV E Protein
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The expression and purification methods for the truncated SARS-CoV E construct corresponding to residues 8–65 (ETR) have been described previously [19 (link)]. This construct does not have cysteines, as these are not required for oligomerization [18 (link),19 (link),28 (link),38 (link)]. In the present work, M9 media was supplemented with an appropriate combination of 15NH4Cl, 13C-glucose, 2H-glucose, and 2H2O (Cambridge Isotope Laboratories) to produce15N-, 13C-, 15N/13C- and 15N/2H-labeled ETR samples. For preparation of fully deuterated 15N/2H-labeled samples, freshly transformed E. coli cells were doubly-selected in LB agar plates and media prepared with 30% and 60% 2H2O, successively, and later grown in M9 media prepared with 99.9% 2H2O [39 (link),40 (link)].
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