Human ubiquitin G76C was expressed in Escherichia coli strain BL21 (DE3) in LB media (Nacalai Tesque) or M9 minimal media containing 99% 15N-labeled ammonium chloride (Cambridge Isotope Laboratories); for the triple-resonance NMR experiments, the M9 medium also contained 99% U-13C-labeled D-glucose (Cambridge Isotope Laboratories). The ubiquitin mutant was purified as described previously5 (link). Protein purity was checked by SDS-PAGE.
15n labeled ammonium chloride
Manufactured by Cambridge Isotopes
Sourced in United States
15N-labeled ammonium chloride is a stable isotope-labeled compound used in scientific research and analysis. It contains the isotope nitrogen-15 (15N) incorporated into the ammonium chloride molecule. This product can be utilized as a tracer or internal standard in various analytical techniques, such as mass spectrometry and nuclear magnetic resonance spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
13c labeled glucose, by Cambridge Isotopes (4 mentions)
Sabouraud dextrose broth, by BD (2 mentions)
Sd agar, by BD (2 mentions)
Chitin column, by New England Biolabs (2 mentions)
Bl21 ai cells, by Thermo Fisher Scientific (2 mentions)
M9 minimal media, by Cambridge Isotopes (2 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Luria bertani media, by Merck Group (1 mentions)
S100 gel filtration column, by GE Healthcare (1 mentions)
Guanidine hydrochloride gdnhcl, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Pet28a, by Merck Group (1 mentions)
Bl21 de3, by Merck Group (1 mentions)
11 protocols using 15n labeled ammonium chloride
1
Recombinant Expression of 15N/13C-Labeled Ubiquitin
2
Overexpression and Purification of Biomolecules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All samples were overexpressed using Escherichia coli BL21(DE3*) cells grown in either Luria-Bertani media (EMD Millipore, Billerica, MA, USA) or M9 minimal media for NMR experiments. Samples for backbone relaxation dispersion were grown in 2H2O-based media with 15N-labeled ammonium chloride (Cambridge Isotopes, Tewksbury, MA, USA).
All samples were purified using an anion-exchange column Q-sepharose (GE Healthcare, Pittsburgh, PA, USA) with buffers A (25 mM HEPES, pH 7.5, 1 mM Na2EDTA) and B (buffer A with 1 M NaCl) using a 0–50% gradient of buffer B. Samples were concentrated with a Corning Spin-X UF centrifugal concentrator (Sigma Aldrich, St. Louis, MO, USA) to a volume of ~1 mL then purified on a S100 gel filtration column (GE Healthcare) in buffer A with 200 mM NaCl.
All samples were purified using an anion-exchange column Q-sepharose (GE Healthcare, Pittsburgh, PA, USA) with buffers A (25 mM HEPES, pH 7.5, 1 mM Na2EDTA) and B (buffer A with 1 M NaCl) using a 0–50% gradient of buffer B. Samples were concentrated with a Corning Spin-X UF centrifugal concentrator (Sigma Aldrich, St. Louis, MO, USA) to a volume of ~1 mL then purified on a S100 gel filtration column (GE Healthcare) in buffer A with 200 mM NaCl.
3
Optimized Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ECOS™ BL21 (DE3) competent Cells were purchased from Yeasterm Biotech Co., Ltd. (Taipei, Taiwan). Lysogeny broth (LB) was purchased from Cyrusbioscience (New Taipei City, Taiwan). Isopropyl β-D-1-thiogalactopyranoside (IPTG), Triton X-100, guanidine hydrochloride (GdnHCl), trifluoroacetic acid, D2O, 2,2-dimethyl-2-siapentane-5-sulfonate (DSS), and ampicillin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 15N-labeled ammonium chloride was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Coomassie Brilliant Blue G-250 was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). Sodium azide was purchased from Merck (Millipore, Burlington, MA, USA).
4
Recombinant Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression vector pET-28a and host BL21 (DE3) were purchased from Novagen (Merck Millipore, Burlington, MA, USA). Restriction enzymes and T4 ligase were purchased from Thermo Scientific (Waltham, MA, USA). LB Broth medium was purchased from MDBio (Taipei, Taiwan), CNBr was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sabouraud Dextrose (SD) Broth and SD agar were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). 15N labeled ammonium chloride was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Other chemicals were from commercial sources.
5
Homology Modeling of Ssa1 and DnaK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. Restriction enzymes were purchased from New England BioLabs (Ipswich, MA). The 13C labeled glucose and 15N labeled ammonium chloride were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Homology modeling of Ssa1 structures in ATP and ADP state were prepared using SWISS-MODEL [88 (link)] and DnaK structures (PDB: 4NJ4 and 2KHO, respectively) as templates. Structural images were prepared with the PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC.
6
Biomolecular Synthesis and Purification
7
Expression and Purification of Ubiquitin Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ubiquitin was expressed using a pET23a-based expression vector in E. coli Rosetta2(DE3) pLysS cells (EMD Millipore) in 2xYT broth by induction with 0.5 mM IPTG and post-induction growth for 4 h at 37°C. Cells were harvested by centrifugation. Purification of the protein was as described elsewhere (24 (link)) with purified protein dialyzed into buffer A (Supplementary Figure S1A). For 15N labeled ubiquitin, the protein was expressed similarly, but in M9 minimal medium supplemented with 15N labeled ammonium chloride (Cambridge Isotope laboratories Inc.) and post-induction growth at 37°C for 14 h.
For PRE experiments, site-directed spin labeling was done at Gly75 of ubiquitin, which was mutated to cysteine. Spin labeling with MTSL and sample preparation was done as described for TDP2 UBA.
Conjugation of monoUb to generate K48- or K63-linked diUbs was done as described elsewhere (24 (link)) with purified conjugated proteins dialyzed into buffer A. Plasmids for all conjugating enzyme expression, pET3d-E2-25K-C170S, pGEX-Ubc13, MBP-His6-TEV-MMS2 and pET21d-Ube1 were obtained from the Addgene plasmid repository. Expression and purification of conjugating enzymes was done as described elsewhere (24 (link)).
For PRE experiments, site-directed spin labeling was done at Gly75 of ubiquitin, which was mutated to cysteine. Spin labeling with MTSL and sample preparation was done as described for TDP2 UBA.
Conjugation of monoUb to generate K48- or K63-linked diUbs was done as described elsewhere (24 (link)) with purified conjugated proteins dialyzed into buffer A. Plasmids for all conjugating enzyme expression, pET3d-E2-25K-C170S, pGEX-Ubc13, MBP-His6-TEV-MMS2 and pET21d-Ube1 were obtained from the Addgene plasmid repository. Expression and purification of conjugating enzymes was done as described elsewhere (24 (link)).
8
Labeling Compounds for Microbial Growth
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
15N-labeled ammonium chloride and 13C-labeled glucose were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Thermanox substrate-coated cover slips were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sabouraud dextrose (SD) broth and SD agar were purchased from Becton, Dickinson, and Company (Franklin Lakes, NJ, USA). All other chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA).
9
Affinity Purification of NpR6012g4 Protein
10
Production and Purification of Isotopically Labeled Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ubiquitin, human 12-kDa FK506-binding protein (FKBP12) and human fatty acid binding protein 4 (FABP4), and their cysteine mutants were expressed in Escherichia coli strain BL21 (DE3) grown in LB or M9 minimal media containing 99% 15N-labeled ammonium chloride (Cambridge Isotope Laboratories). Human ubiquitin was purified by cation exchange and size-exclusion chromatography. Human FKBP12 was expressed as a fusion protein with an N-terminal glutathione S-transferase and small ubiquitin-like modifier protein (GST-SUMO-1) tag. After cleavage of the tag by GST-SENP2 protease, FKBP12 was further purified by size-exclusion chromatography. Human FABP4 was expressed as a fusion protein with an N-terminal hexa-histidine (His6) SUMO-1 protein tag. After cleavage of the His6-SUMO tag by GST-SENP2 protease, FABP4 was further purified by size-exclusion chromatography. N-terminally ubiquitylated FKBP12 and FABP4 were expressed as fusion proteins with an HRV3C-cleavable C-terminal His6-tag and purified by Ni-NTA affinity chromatography. After cleavage of the C-terminal His6-tag by HRV3C protease, the proteins were further purified by anion exchange and size-exclusion chromatography.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!