CNCbl and AdoCbl were purchased from MilliporeSigma. CNpCbl was extracted from Propionibacterium acidi-propionici strain DSM 20273 and purified as described (59 (link), 60 (link)). AdopCbl was chemically synthesized from CNpCbl and purified as described (16 (link)). Cobamides were quantified spectrophotometrically (16 (link), 22 ). Cbl and pCbl were used in their cyano forms (CNCbl and CNpCbl) unless otherwise indicated.
Cncbl
Manufactured by Merck Group
Sourced in Germany
CNCbl is a laboratory instrument designed for the precise and automated control of various manufacturing processes. It utilizes computer numerical control (CNC) technology to execute complex operations with high accuracy and repeatability. The core function of CNCbl is to provide precise, programmable control over the movement and operation of machinery used in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Adocbl, by Merck Group (3 mentions)
Porcine gastric mucin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by PanEco (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Phenformin, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Sodium acetate trihydrate, by Merck Group (1 mentions)
Glacial acetic acid hplc grade, by Avantor (1 mentions)
Ohcbl hcl, by Merck Group (1 mentions)
Mecbl, by Merck Group (1 mentions)
Sodium cyanide, by Merck Group (1 mentions)
Lc ms grade methanol, by Honeywell (1 mentions)
Adenosylcobalamin, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Laboratory reagent grade, by Thermo Fisher Scientific (1 mentions)
Analytical reagent grade, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Merck Group (1 mentions)
6 protocols using cncbl
1
Cobamide Extraction and Quantification
2
Porcine Gastric Mucin and Coenzyme B12 Protocols
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Porcine gastric mucin was purchased from MilliporeSigma (M1778). AdoCbl (coenzyme B12), MeCbl, CNCbl, and (CN)2Cbi were purchased from MilliporeSigma.
3
Analytical Reagents for Biochemical Assays
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Horseradish peroxidase (HRP), GSH, NAC, DTT, AA, 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol), HOCbl, CNCbl, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was from Gibco (Carlsbad, CA, USA). DDC was purchased from MP Biomedicals (Irvine, CA, USA). Phosphate buffered saline (PBS) was obtained from Paneco (Moscow, Russia). All reagents were of analytical grade purity. Water used for the preparation of solutions was purified USING a Milli-Q system (Millipore, Burlington, MA, USA).
4
Assessing Biguanide Treatment Effects
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CN-Cbl (Sigma Aldrich, Cat# V900445) was dissolved in ddH2O as a stock solution at 15 mM. Metformin and phenformin (Sigma Aldrich, Cat# D150959 and Cat# PHR157) were prepared in ddH2O as stock solutions at 1 M and 0.2 M, respectively. Varied doses of biguanides were applied in this study according to previous studies18 (link),38 (link) and the level of tolerance of tested organisms to the drugs. In brief, 50 mM Metformin was used in the on-plate treatment for ∆nhr-114 worms, and 200 mM was used in all bacterial treatments. The same volumes of ddH2O were used as vehicle (Veh). phenformin at 4 mM was used in all bacterial treatments for feeding Pacdh-1::GFP worms, LC-MS/MS measurement, growth measurement and qPCR. phenformin was used at an optimized concentration, 2 mM, in the Keio Collection screen for feeding Δnhr-114 worms to ensure sufficient bacterial growth. The highest dose of phenformin (5 mM) was used in on-plate treatments or growth inhibition assays to screen for the strongest phenformin-resistant strains. All reagents were filtered with 0.22 μm membranes prior to use and stored at −20 °C.
5
Synthesis and Characterization of Labeled Cobalamins
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Analytical standards of CNCbl, OHCbl·HCl, AdoCbl, and MeCbl were obtained from Sigma-Aldrich (Steinheim, Germany). [15N13]-CNCbl was prepared in-house as previously described [8 (link)]. In general, 15N-labeled cobalamins were prepared via biosynthesis using Propionibacterium freudenreichii. The labeled cobalamins were either directly isolated from the cell extracts or further treated with additional chemical modification steps to obtain different vitamers. Papain from papaya latex (EC 3.4.22.2, cat no. P4762, ≥ 10 units/mg protein), α-amylase from Aspergillus oryzae (EC 3.2.1.1, cat. no. 10065, ~30 U/mg), and soybean flour (type I, cat. no. S9633) were purchased from Sigma-Aldrich (Steinheim, Germany).
Sodium cyanide (≥ 97%) and sodium acetate trihydrate (≥ 99.0%) were purchased from Sigma-Aldrich (Steinheim, Germany). Water (LC-MS and HPLC grade) and methanol (HPLC grade) were obtained from Th. Geyer (Renningen, Germany). Acetic acid for LC-MS (≥ 99.8%), glacial acetic acid (HPLC grade), and ammonium acetate for LC-MS (≥ 99.0%) were purchased from VWR International (Ismaning, Germany). Methanol (LC-MS grade) was purchased from Honeywell (Seelze, Germany).
Sodium cyanide (≥ 97%) and sodium acetate trihydrate (≥ 99.0%) were purchased from Sigma-Aldrich (Steinheim, Germany). Water (LC-MS and HPLC grade) and methanol (HPLC grade) were obtained from Th. Geyer (Renningen, Germany). Acetic acid for LC-MS (≥ 99.8%), glacial acetic acid (HPLC grade), and ammonium acetate for LC-MS (≥ 99.0%) were purchased from VWR International (Ismaning, Germany). Methanol (LC-MS grade) was purchased from Honeywell (Seelze, Germany).
6
Quantification of Cobalamin Compounds by HPLC-ICP-MS
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The different cobalamin (Cbl) standards, Methyl-cobalamin (Me-Cbl), Hydroxycobalamin (HO-Cbl), Adenosyl-cobalamin (Ado-Abl) and cyano-cobalamin (CN-Cbl) (98%, Sigma-Aldrich Germany) were dissolved in water with a concentration of 1 mg Co/mL and stored in the dark. The eluents for the HPLC were 0.1% formic acid (Analytical reagent grade, Fisher Scientific UK) in water (eluent A) and 0.1% formic acid in methanol (HPLC grade S, Rathburn UK) (eluent B). Co standards (High purity standards, UK) for calibration were prepared based on a stock solution of 1000 mg/L diluted with 1% HNO3, conc. (supra pure, BDH UK). A Rh solution (Specpure, Alfa Aesar Germany) served as internal standard. For the sample preparation different organic solvents were used, including methanol (Laboratory reagent grade, Fisher Scientific UK), acetone (Laboratory reagent grade, Fisher Scientific UK), and additionally ultra-pure water (18 MΩ cm). For the liver extraction acetate buffer (pH 5) (acetic acid: extra pure, Sigma-Aldrich Germany), Papain (from Carica Papaya, Sigma Aldrich Germany), potassium cyanide (Fisher Scientific UK) and HCl (Laboratory reagent grade, Fisher Scientific UK) were used. Nitric acid conc and hydrogen peroxide (Laboratory reagent grade, Fisher Scientific UK) were used for the microwave-assisted digestion of liver samples prior to total Co measurements by ICP-MS.
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