Chitin from shrimp shells (practical grade coarse flakes), chitosan CHIT50.1 (Product number: 900,344) and CHIT50.2 (Product number: 448,869), N-acetyl-D-glucosamine (GlcNAc) and biotin were from Sigma Aldrich (St. Louis, MO, USA). Chitosan from shrimp shells CHIT100 and CHIT600 were from Across Organics Chemicals (ThermoFisher Scientific, Waltham, Massachusetts, USA). Colloidal chitin (CC) was obtained as referred previously [30 (link)]. Basically, 10 g chitin in 10 M HCl was maintained 16 h at 4 ºC, filtered through glass thick fibres into ethanol and then chitin floccules were precipitated at 4 ºC, collected (5000xg during 10 min) and maintained in 0.1 M sodium acetate pH 4.0-4.5. Chitosan was dissolved in 0.1 M sodium acetate pH 4.0-4.5, in a final concentration of 10 g l− 1. Diacetyl-chitobiose ((GlcNAc)2), triacetyl-chitotriose ((GlcNAc)3), tetraacetyl-chitotetraose ((GlcNAc)4), pentaacetyl-chitopentaose ((GlcNAc)5) and hexaacetyl-chitohexaose ((GlcNAc)6) were from Megazyme (Bray, Irland). Nitrogen base w/o amino acids (YNB) was from Difco (BD, Sparks, MD, USA). Yeast extract, peptone, tryptone and agar were from Laboratorios Conda S.A. (Madrid, Spain).
N acetyl d glucosamine glcnac
Manufactured by Merck Group
Sourced in United States
N-acetyl-D-glucosamine (GlcNAc) is a monosaccharide that is a derivative of glucose. It is a key component of the biopolymer chitin, which is found in the exoskeletons of arthropods and the cell walls of fungi. GlcNAc is also involved in various biological processes, including cell signaling and glycosylation of proteins.
Automatically generated - may contain errors
Lab products found in correlation
D mannose, by Merck Group (3 mentions)
D galactose, by Merck Group (3 mentions)
D glucose, by Merck Group (3 mentions)
N acetyl d galactosamine galnac, by Merck Group (3 mentions)
L fucose, by Merck Group (2 mentions)
D allose, by Merck Group (2 mentions)
L rhamnose, by Merck Group (2 mentions)
D xylose, by Merck Group (2 mentions)
D glucuronic acid glca, by Merck Group (2 mentions)
D galacturonic acid gala, by Merck Group (2 mentions)
L arabinose, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
D fructose, by Merck Group (2 mentions)
Glacial acetic acid, by Merck Group (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Biotin, by Merck Group (1 mentions)
Diacetyl chitobiose, by Megazyme (1 mentions)
Pentaacetyl chitopentaose, by Megazyme (1 mentions)
Hexaacetyl chitohexaose, by Megazyme (1 mentions)
Microcal itc200 system, by Malvern Panalytical (1 mentions)
9 protocols using n acetyl d glucosamine glcnac
1
Chitin and Chitosan Characterization
2
Thermodynamic Profiling of YwfG Variants
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A MicroCal iTC200 system (Malvern Panalytical, Almelo, Netherlands) was used to measure enthalpy changes associated with interactions between the YwfG variants and the following yeast cell-wall components: the monosaccharides d -glucose, d -galactose, d -mannose, d -fucose, N-acetyl-d -glucosamine (GlcNAc), and 2-(acetylamino)-2-deoxy-d -galactose (GalNAc) (Sigma-Aldrich); α-1,2-, α-1,3-, α-1,4-, and α-1,6-mannobiose (Dextra Laboratories, Reading, UK); mannan from S. cerevisiae (Sigma-Aldrich); and yeast mannoproteins.
A solution of YwfG variant (100 μM YwfG28–270, YwfG28–336, YwfG28–511, or MubR4 in 20 mM Tris-HCl [pH 7.5]) was placed in a 200-μL calorimeter cell, and test solution (10 mM for monosaccharides, 2 mM for monosaccharides and mannobioses, 10 mg mL−1 for yeast mannan, OD280 1.0 for mannoproteins) was loaded into the injection syringe. The test solution was titrated into the sample cell as a sequence of 20 injections of 2-μL aliquots each. All experiments were performed at 25°C.
A solution of YwfG variant (100 μM YwfG28–270, YwfG28–336, YwfG28–511, or MubR4 in 20 mM Tris-HCl [pH 7.5]) was placed in a 200-μL calorimeter cell, and test solution (10 mM for monosaccharides, 2 mM for monosaccharides and mannobioses, 10 mg mL−1 for yeast mannan, OD280 1.0 for mannoproteins) was loaded into the injection syringe. The test solution was titrated into the sample cell as a sequence of 20 injections of 2-μL aliquots each. All experiments were performed at 25°C.
3
Comprehensive Carbohydrate Analysis Protocol
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All foods and food products were purchased from local markets (Davis and Sacramento, CA, USA) including Safeway, Trader Joe’s, Davis Food Co-op, Whole Foods, Nugget Markets, Target, and online (Amazon). Trifluoroacetic acid (TFA, HPLC grade), 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP), chloroform (HPLC grade), ammonium hydroxide solution (NH4OH) (28–30%), ammonium acetate, sodium acetate, glacial acetic acid, methanol (HPLC grade), D-fructose, D-mannose, D-allose, D-glucose, D-galactose, L-rhamnose, L-fucose, D-ribose, D-xylose, L-arabinose, N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), D-glucuronic acid (GlcA), and D-galacturonic acid (GalA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arabinoxylan and polygalacturonic acid were purchased from Megazyme (Bray, Ireland). 96-well Nunc plates and lids were purchased from Thermo Scientific. Viscozyme was provided by Novozyme (Davis, CA, USA). Acetonitrile (ACN) (HPLC grade) was purchased from Honeywell (Muskegon, MI, USA). Nanopure water was used for all experiments.
4
Comprehensive Glycan Analysis Protocol
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Sodium acetate, hydrogen peroxide (H2O2) (30% v/v), iron(III)
sulfate pentahydrate (Fe2(SO4)3), trifluoroacetic acid
(TFA) (HPLC grade), chloroform (HPLC grade), ammonium acetate, sodium hydroxide pellets
(semiconductor grade 99.99% trace metals basis), ammonium hydroxide solution
(NH4OH) (28–30%), 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP),
dichloromethane (DCM), methanol (HPLC grade), anhydrous dimethyl sulfoxide (DMSO), glacial
acetic acid,d -galactose, d -mannose, d -glucose,
d -allose, d -fructose, d -xylose, l -arabinose,
d -xylose, l -fucose, l -rhamnose,
N-acetyl-d -glucosamine (GlcNAc),
N-acetyl-d -galactosamine (GalNAc), d -glucuronic acid
(GlcA), andd -galacturonic acid (GalA) were purchased from Sigma-Aldrich (St.
Louis, MO). Maltotriose, maltotetraose, maltopentaose, and maltohexaose were obtained from
Carbosynth (Compton, UK). Galactan was purchased from Megazyme (Bray, Ireland). C18 and
porous graphitized carbon (PGC) solid phase extraction (SPE) plates were purchased from
Glygen (Columbia, MD). Formic acid (FA) (99.5% optima LC/MS grade) was purchased from
Fisher Scientific (Hampton, NH). Acetonitrile (ACN) (HPLC grade) was purchased from
Honeywell (Muskegon, MI). All purchased chemicals were used without further purification.
Butternut squash was purchased from Pedrick Produce (Dixon, CA). Nanopure water was used
for all experiments.
sulfate pentahydrate (Fe2(SO4)3), trifluoroacetic acid
(TFA) (HPLC grade), chloroform (HPLC grade), ammonium acetate, sodium hydroxide pellets
(semiconductor grade 99.99% trace metals basis), ammonium hydroxide solution
(NH4OH) (28–30%), 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP),
dichloromethane (DCM), methanol (HPLC grade), anhydrous dimethyl sulfoxide (DMSO), glacial
acetic acid,
N-acetyl-
N-acetyl-
(GlcA), and
Louis, MO). Maltotriose, maltotetraose, maltopentaose, and maltohexaose were obtained from
Carbosynth (Compton, UK). Galactan was purchased from Megazyme (Bray, Ireland). C18 and
porous graphitized carbon (PGC) solid phase extraction (SPE) plates were purchased from
Glygen (Columbia, MD). Formic acid (FA) (99.5% optima LC/MS grade) was purchased from
Fisher Scientific (Hampton, NH). Acetonitrile (ACN) (HPLC grade) was purchased from
Honeywell (Muskegon, MI). All purchased chemicals were used without further purification.
Butternut squash was purchased from Pedrick Produce (Dixon, CA). Nanopure water was used
for all experiments.
5
Gametocyte Enrichment and PF-543 Treatment
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Tightly synchronized PfRKL-9 parasites were treated with N-Acetyl-d-Glucosamine (GlcNAc, Sigma-Aldrich) for 72 h to eliminate the asexual stage parasites. The synchronized gametocyte culture (typically at 2% to 4% gametocytaemia), thus obtained, was diluted to 1% hematocrit and seeded (100 μL per well) in 96-well flat-bottom plates (Corning,) in the presence of PF-543 at its EC50 (172 nM) and 2× EC50 values. Untreated gametocytes served as control. The culture was maintained for 12 days in complete media containing the appropriate concentrations of PF-543. Gametocytaemia was monitored daily by microscopic examination of Giemsa-stained thin blood smears, and the percent gametocytaemia was calculated using the following formula, % gametocytaemia = (Number of gametocyte /Total number of cells) ×100. The graph was plotted using GraphPad Prism version 8.0.
6
Chitin-Derived Oligosaccharide Generation
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Mixtures containing 0.036 mg/ml of ChiA74Δsp, 0.5% (w/v) colloidal chitin, and 20 mM phosphate buffer (pH 7.0) were incubated at 37 °C, 200 rpm for 12 h to generate chitin-derived oligosaccharides (OGS). Reaction was terminated by heating at 100 °C for 5 min. Samples were centrifuged, supernatants were collected and concentrated in a DNA120 SpeedVac (ThermoSavant), then pellets were resuspended in 10 µl of double distilled water and 10 µl of methanol. OGS were analyzed by silica gel (Merk) thin layer chromatography (TLC) using 5:4:3 (v/v/v) n-butanol: methanol:16% aqueous ammonia as the mobile phase. Samples were visualized by staining with 20% (v/v) sulfuric acid in ethanol and incubated at 80 °C until chitin-derived oligosaccharides signals were developed. Molecular markers (Sigma, St. Louis, MO) N-acetyl-D-glucosamine (GlcNAc) and N,N′-diacetylchitobiose (GlcNAc2) were used to determine the degree of polymerization (DP)22 (link).
7
Glycan Analysis of Porcine Mucin
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Calcofluor white M2R (Calcofluor; F3543), N-Acetyl-D-glucosamine (GlcNAc; A4106), N-Acetyl-D-galactosamine (GalNAc; A2795), D-(+)-Fucose (Fucose; F8150), D-(+)-Mannose (Mannose; M6020), Mucin from porcine stomach (Porcine mucin; M2378) and WGA-FITC conjugate (L4895) were all purchased from Sigma-Aldrich (Lyon, France).
8
DRG Myelination Assay with GlcNAc
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DRG explants were isolated from E13.5 embryos, seeded on rat collagen I-coated coverslips and maintained in culture as previously described [77 (link)]. Myelination was induced with 50μg/ml ascorbic acid (Sigma-Aldrich) added to culture medium. Treatments with N-acetyl-D-Glucosamine (GlcNAc; Sigma-Aldrich), dissolved in culture medium, were performed for 2 or 3 weeks in parallel to myelination induction. Culture medium was refreshed every two days. Samples were fixed and prepared for immunofluorescence and the average number and length of MBP+ internodes per field were measured with NIH Image-J software. 8 non-overlapping images per DRG were acquired with a Leica DM5000 microscope (10x and 20x objectives) equipped with a Leica DFC480 digital camera. At least 3 independent dissections were performed.
9
Raman Optical Activity of Carbohydrates
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N-acetyl-D-glucosamine (GlcNAc) and the sodium salt of Dglucuronic acid (GlcA) were purchased from Sigma-Aldrich and used without further purification. The ROA spectra were measured at ambient temperature in water using the previously described ChiralRAMAN instrument (BioTools, Inc.), 48 which employs the scattered circular polarization (SCP) measurement strategy. The ROA spectra are presented as circular intensity differences (I R -I L ) with I R and I L denoting the Raman intensities with right-and left-circular polarization, respectively. The sample concentrations were ~150 mM for each sample, with pH values of 7.0 and 6.3 for GlcA and GlcNAc, respectively. Experimental conditions: laser wavelength 532 nm; laser power at the sample 350 mW; spectral resolution 7 cm -1 ; acquisition times 17-18 hrs. Cosmic ray spikes were removed from the ROA spectra by means of a median filter, after which the spectra were smoothed using a second-order Savitzky-Golay filter.
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