15n nh4cl

Manufactured by Merck Group

15N-NH4Cl is a laboratory reagent that serves as a stable isotope-labeled compound. It contains the isotope nitrogen-15 (15N) bound to ammonium chloride (NH4Cl). This product is commonly used as a tracer in various scientific applications that require the monitoring or quantification of nitrogen-containing compounds.

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15N‐labeled NH4Cl NH4Cl

6 protocols using 15n nh4cl

Recombinant 15N-Tau Protein Production and Purification

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pET15b-Tau recombinant T7lac expression plasmid was transformed into competent E. coli BL21 (DE3) bacterial cells. A small-scale culture was grown in LB medium at 37°C and was added at 1:10 v/v to 1 L of a modified M9 medium containing MEM vitamin mix 1X (Sigma-Aldrich), 4 g of glucose, 1 g of ¹⁵N-NH₄Cl (Sigma-Aldrich), 0.5 g of ¹⁵N-enriched Isogro (Sigma-Aldrich), 0.1 mM CaCl₂, and 2 mM MgSO₄. Recombinant ¹⁵N Tau production was induced with 0.5 mM IPTG when the culture reached an optical density at 600 nm of 0.8. Proteins were first purified by heating the bacterial extract, obtained in 50 mM NaPi, pH 6.5, 2.5 mM EDTA, and supplemented with complete protease inhibitor cocktail (Sigma-Aldrich), 15 min at 75°C. The resulting supernatant was next passed on a cation exchange chromatography column (Hitrap SP sepharose FF, 5 mL, Cytiva) with 50 mM NaPi, pH 6.5, and eluted with a NaCl gradient. Tau proteins were buffer-exchanged against 50 mM ammonium bicarbonate (Hiload 16/60 desalting column, Cytiva) for lyophilization. The same protocol⁶⁹ was used to produce and purify Tau 2N3R isoform, Tau[244–368] (designated MTBD, also called K18 fragment), chimeric Tau[244–368] with two PHF6 or PHF6∗ peptide sequences instead of PHF6∗ and PHF6 sequences (Figure S10A) and Tau [208–324].

Danis C., Dupré E., Zejneli O., Caillierez R., Arrial A., Bégard S., Mortelecque J., Eddarkaoui S., Loyens A., Cantrelle F.X., Hanoulle X., Rain J.C., Colin M., Buée L, & Landrieu I. (2022). Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment. Molecular Therapy, 30(4), 1484-1499.

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Synthesis and Characterization of Modified Nucleosides

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All salts were obtained from Sigma Aldrich (Munich, Germany) at molecular biology grade unless stated otherwise. Isotopically labeled compounds: ¹⁵N-NH₄Cl (≥98% atom, Sigma-Aldrich). ¹³C₆-glucose (≥99% atom, Sigma-Aldrich) and l-methionine-methyl-D₃ (98 atom % D, Sigma-Aldrich). All solutions and buffers were made with water from a Millipore device (Milli-Q, Merck, Kenilworth, NJ, USA).
Supplementary Materials Table S1 shows all synthetic standards of modified nucleosides and their respective vendors.

Borland K., Diesend J., Ito-Kureha T., Heissmeyer V., Hammann C., Buck A.H., Michalakis S, & Kellner S. (2019). Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis. Genes, 10(1), 26.

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Purification of Recombinant Proteins

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Restriction endonucleases, DNA ligase, and calf intestinal alkaline phosphatase were obtained from New England Biolabs (MA). Isopropyl β-d-thiogalactoside (IPTG), acrylamide, bisacrylamide, and Tris(2-carboxyethyl)phosphine hydrochloride were purchased from Sigma Chemical Company (St. Louis, MO). Diethylaminoethyl (DEAE) cellulose (DE52) was from Whatman Laboratories. Shodex carboxymethyl cellulose HPLC preparative column (CM 2025) was from Phenomenex. YM10 membrane filters were from EMD Millipore Corporation (Bedford, MA). Ni-Sepharose column HisTrap HP and MonoQ anion-exchange column were obtained from GE Healthcare, Bio-Science (Uppsala, Sweden). BugBuster protein extraction reagent was from Millipore (Billerica, MA). Protease inhibitor cocktail was purchased from Roche (Indianapolis, IN). Benzonase nuclease was from Novagen (Denmark). ¹⁵N-NH₄Cl was obtained from Sigma Chemical Company (St. Louis, MO).

Reddy P.T., Jaruga P., Nelson B.C., Lowenthal M.S., Jemth A.S., Loseva O., Coskun E., Helleday T, & Dizdaroglu M. (2015). Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements. Methods in enzymology, 566, 305-332.

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Recombinant 15N-labeled Tau Protein Production

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pET15b-tau recombinant T7lac expression plasmid was transformed into competent E. coli BL21 (DE3) bacterial cells. A small-scale culture was grown in LB medium at 37 °C and was added at 1:10 V/V to 1 L of a modified M9 medium containing MEM vitamin mix 1× (Sigma-Aldrich), 4 g of glucose, 1 g of ¹⁵N-NH₄Cl (Sigma-Aldrich), 0.5 g of ¹⁵N-enriched Isogro (Sigma-Aldrich), 0.1 mM CaCl_2, and 2 mM MgSO₄. Recombinant ¹⁵N tau (NCBI reference number NP_005901.2) production was induced with 0.5 mM IPTG when the culture reached an optical density at 600 nm of 0.8. Proteins were first purified by heating the bacterial extract, obtained in 50 mM NaPi pH 6.5, 2.5 mM EDTA, and supplemented with complete protease inhibitors cocktail (Sigma-Aldrich), 15 min at 75 °C. The resulting supernatant was next passed on a cation exchange chromatography column (Hitrap SP sepharose FF, 5 ml, Cytiva) with 50 mM NaPi pH 6.5 and eluted with a NaCl gradient. Tau proteins were buffer-exchanged against 50 mM ammonium bicarbonate (Hiload 16/60 desalting column, Cytiva) for lyophilization. Detailed procedure can be found in (62 ).
K18 expression and purification were performed according to (63 (link)).

Mortelecque J., Zejneli O., Bégard S., Simões M.C., ElHajjar L., Nguyen M., Cantrelle F.X., Hanoulle X., Rain J.C., Colin M., Gomes C.M., Buée L., Landrieu I., Danis C, & Dupré E. (2024). A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein. The Journal of Biological Chemistry, 300(4), 107163.

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Recombinant Production of Isotope-Labeled SH3c Domain

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The ¹⁵N-labeled SH3c domain of hCIN85 was recombinantly produced in Toronto minimal medium with ¹⁵N-NH₄Cl (Sigma Aldrich) as nitrogen source according to a published protocol^{42 (link)}. Briefly, a fragment of CIN85 comprising amino acids 263-333 was expressed in the bacterial strain BL21(DE3)Star (Invitrogen) as fusion protein with N-terminal His₇-tag. After purification on a Ni-NTA Protino^TM metal affinity column (Macherey-Nagel, Germany) the His₇-tag was cleaved off with TEV-protease and removed by a second Ni-NTA Protino^TM column purification step. The SH3c (this construct is referred to as SH3c in the main manuscript) domain was eluted in the flow-through and further purified by gel-filtration on a Superdex 75/16-60 column (GE Healthcare). The sample was dialyzed against NMR buffer (20 mM sodium phosphate, pH 6.5, 100 mM NaCl, 10 mM TCEP, 0.05% (w/v) NaN₃) and the final concentration was adjusted to 2 mM.

Chakrabarti K.S., Olsson S., Pratihar S., Giller K., Overkamp K., Lee K.O., Gapsys V., Ryu K.S., de Groot B.L., Noé F., Becker S., Lee D., Weikl T.R, & Griesinger C. (2022). A litmus test for classifying recognition mechanisms of transiently binding proteins. Nature Communications, 13, 3792.

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Preparing Lipids and Proteins for Cell Wall Analysis

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[¹³C]‐glucose was purchased from Cambridge Isotope Labs. The [¹⁵N]‐NH₄Cl, and all other chemicals used, were purchased from Sigma‐Aldrich unless otherwise stated. Lipid II versions were prepared as previously described (Breukink et al., 2003; Bertsche et al., 2005). The following proteins and antibodies were prepared as previously described: LpoB (Egan et al., 2014), UB2H (Egan et al., 2014), CpoB (Gray et al., 2015), and anti‐PBP1B (Bertsche et al., 2006). CpoB^TPR and CpoB^CC versions were prepared by the same procedure as for CpoB. Cellosyl was provided by Hoechst AG, Frankfurt (Germany). Bacillus cereus β‐lactamase (569/H9) was purchased from Merck.

Egan A.J., Maya‐Martinez R., Ayala I., Bougault C.M., Banzhaf M., Breukink E., Vollmer W, & Simorre J. (2018). Induced conformational changes activate the peptidoglycan synthase PBP1B. Molecular Microbiology, 110(3), 335-356.

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