Fluorescein isothiocyanate (FITC) used as analyte was supplied by Sigma-Aldrich (St. Louis, MO, USA). It was dissolved in an aqueous electrolyte of a selected pH at a final concentration of 50 ng∙mL−1 (50 ppb) or 1 µg∙mL−1 (1 ppm). In the MST method, it was manually introduced into the MST capillaries by immersing them in the solution. In measurements conducted with the CE-MST system, 2-aminoacridone (AMAC) was used as intentionally added interferent (Sigma-Aldrich), its final concentration was 50 µg∙mL−1 (50 ppm). For the experiments involving the commercial CE instrument, two sets of electrolytes were prepared covering a wide range of pH, 4–9, based on phosphate (NaH2PO4/Na2HPO4) and acetate (CH3COOH/CH3COONa) buffers (Sigma-Aldrich). It was decided to cross the buffering range in order to maintain the consistency of ionic composition throughout the tested pH range. The buffers were selected in such a way that their buffering capabilities complemented each other (acetate up to pH 6, phosphate over pH 6). Their ionic strength was 50 ± 10 mM. A different set of electrolytes was used in the case of portable CE setup (see Section 3.5 ). All other reagents were supplied by Avantor Performance Materials Poland. SA (Gliwice, Poland).
2 aminoacridone amac
Manufactured by Merck Group
Sourced in United States
2-aminoacridone (AMAC) is a fluorescent dye used as a labeling reagent in analytical chemistry and biochemistry. It is a heterocyclic aromatic compound that can be used to derivatize carbohydrates, glycoproteins, and other biomolecules for detection and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Sodium cyanoborohydride, by Merck Group (3 mentions)
Sodium cyanoborohydride nacnbh3, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
δua glcnac, by Iduron (2 mentions)
4 deoxy α l threo hex 4 enopyranosyluronic acid, by Iduron (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Deae sepharose fast flow resin, by GE Healthcare (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Diethylaminoethyl deae sepharose fast flow resin, by GE Healthcare (1 mentions)
Ltq velos pro, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000rs hplc, by Thermo Fisher Scientific (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
D glucuronic acid, by Merck Group (1 mentions)
Prostaglandin e1, by Merck Group (1 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (1 mentions)
Tnf α, by Sino Biological (1 mentions)
10 protocols using 2 aminoacridone amac
1
FITC Quantification via MST and CE
2
Fluorescent Labeling of Biomolecules
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2-Aminoacridone (AMAC) (Sigma-Aldrich 06627) and sodium cyanoborohydride (NaCNBH3) (Sigma-Aldrich 156159) Calcium chloride (449709), DMSO (Sigma-Aldrich 94563), acetic acid (Sigma-Aldrich A6283), ammonium acetate (Sigma-Aldrich 73594) and methanol (Sigma-Aldrich 646377) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3
Comparative Analysis of Bat and Human Lung Glycosaminoglycans
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Eight bat lungs (4 male and 4 female) were harvested from rabies negative wild Big Brown bats (Eptesicus fuscus) submitted for routine rabies testing. Tissue was autoclaved to ensure any other potential pathogens that may be present in these bats would be rendered non-infectious. Previous studies have demonstrated that GAGs are stable under standard conditions used in autoclaving; indeed, heparin is routinely sterilized by autoclaving. Additionally, 6 human lung tissue samples derived from male cadavers were obtained from the University of Vermont (UVM) autopsy services under appropriate institutional guidelines. Porcine intestinal heparin (∼18 kDa) was from Celsus (Cincinnati, OH, USA). 4-deoxy-α-l -threo-hex-4-enopyranosyluronic acid (ΔUA)-N-acetyl-d -glucosamine (GlcNAc) (HS 0S); ΔUA-N-sulfo-d -glucosamine (GlcNS) (HS NS); ΔUA-GlcNAc6S (HS 6S); ΔUA2S-GlcNAc (HS 2S); ΔUA-GlcNS6S (HS NS6S); ΔUA2S-GlcNS (HS NS2S); ΔUA2S-GlcNAc6S (HS 2S6S); ΔUA2S-GlcNS6S (HS TriS); ΔUA-N-acetyl-d -galactosamine (GalNAc) (CS 0S); ΔUA2S-GalNAc (CS 2S); ΔUA-GalNAc4S (CS 4S); ΔUA-GalNAc6S (CS 6S); ΔUA2S-GalNAc4S (CS 2S4S); ΔUA2S-GalNAc6S (CS 2S6S); ΔUA-GalNAc4S6S (CS 4S6S); ΔUA2S-GalNAc4S6S (CS TriS); ΔUA-GlcNAc (hyaluronan, HA) were purchased from Iduron (Manchester, UK). 2-aminoacridone (AMAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Actinase E was from Kaken Biochemicals (Tokyo, Japan).
4
Heparan Sulfate Disaccharide Analysis in Mice
5
Glycosylation Analysis of HIV-1 Envelope Glycoproteins
6
Heparan Sulfate Quantification Workflow
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2-Aminoacridone (AMAC) and sodium cyanoborohydride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Four 13C-labeled 8-mer calibrants and one 13C-labeled 10-mer calibrant were synthesized by chemoenzymatic synthesis method (19 (link)). Recombinant heparan lyase I and II were expressed in Escherichia coli and purified by a Ni-agarose column. Diethylaminoethyl (DEAE) Sepharose Fast Flow resin was purchased from GE Healthcare (Chicago, IL, USA). All reagents and chemicals were HPLC grade or LC-MS grade.
7
AMAC Labeling and MS/MS Analysis of Chitooligosaccharides
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Products generated by the deacetylase from (GlcNAc)5 were labeled with 2-aminoacridone (AMAC) (Sigma- Aldrich, Steinheim, Germany) as previously described by Bahrke et al. [30 (link)] and labeled products were purified using a C18 column (Starata C18E, Phenomenex, CA, US) as described by Morelle et al. [31 (link)], with one deviation: instead of lyophilizing the labeled samples, the reaction products were dried by vacuum centrifugation. The labeled products were re-dissolved in 50 μl 50% MeOH and analyzed using a LTQ-Velos Pro ion trap mass spectrometer (Thermo Scientific, Bremen, Germany) connected to an Ultimate 3000 RS HPLC (Dionex, CA, USA). This setup was used for direct injection without a column. The pump delivered 200 μl/min of 0.03 μM formic acid in 70% acetonitrile and data was acquired for 24 seconds after injection. For the MS, the capillary voltage was set to 3.5 kV and the scan range was m/z 150–2000 using two micro scans. The automatic gain control was set to 10,000 charges and a maximum injection time of 20 milliseconds. For fragmentation of desired precursor masses by MS2, the normalized collision energy was set to 37 and three micro scans were used. The data were recorded with Xcalibur version 2.2.
8
Glycosaminoglycan Analysis in Carcinoma
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Carcinoma tissues were lyophilized. Glycosaminoglycan preparation, lyase treatment, fluorescence disaccharide labeling and separation were performed according to [31 (link)]. Briefly, tumors were protease- and DNAase digested, and GAGs were purified on anion-exchange chromatography. Then, 500ng GAGs, as estimated by the carbazol method, were subjected to degradation by chondroitinase ABC (Sigma) or by a mixture of heparinases (in-house preparation, purified from E.coli transformed with the pET-15b expression vector carrying heparinase I, or pET-19b expression vector carrying heparinase II or III; provided by prof. Jian Liu, University of North Carolina). Fluorophore-labeling of the resulting disaccharides was performed by 2-aminoacridone (AMAC, Sigma). Pre-column AMAC-labeled disaccharides were analyzed by HPLC as described previously [31 (link)]. Quantification was done by comparison to known weight of mock-treated standard disaccharides (Iduron, UK).
9
Glycosaminoglycan Disaccharide Standards Analysis
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Unsaturated disaccharide standards of chondroitin sulfate (CS; ΔUA-GalNAc; ΔUA-GalNAc4S; ΔUA-GalNAc6S; ΔUA2S-GalNAc; ΔUA2S-Gal-NAc4S; ΔUA2S-GalNAc6S; ΔUA-GalNAc4S6S; ΔUA2S-GalNAc4S6S), unsaturated disaccharide standards of heparin sulfate (HS; ΔUA-GlcNAc; ΔUA-GlcNS; ΔUA-GlcNAc6S; ΔUA2S-GlcNAc; ΔUA2S-GlcNS; ΔUA-GlcNS6S; ΔUA2S-GlcNAc6S; ΔUA2S-GlcNS6S), and unsaturated disaccharide standard of hyaluronic acid (HA; ΔUA-GlcNAc), where ΔUA is 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid, were purchased from Iduron. Actinase E was obtained from Kaken Biochemicals. Chondroitin lyase ABC from Proteus vulgaris was expressed in the laboratory of R.J.L. Recombinant flavobacterial heparin lyases I, II, and III were expressed in the R.J.L. laboratory using Escherichia coli strains provided by Jian Liu (College of Pharmacy, University of North Carolina, Chapel Hill, NC). 2-aminoacridone (AMAC) and sodium cyanoborohydride (NaCNBH3) were obtained from Sigma-Aldrich. All other chemicals were of HPLC grade. Vivapure Q Mini H strong anion exchange spin columns were from Sartorius.
10
Assessing Endothelial Cell Barrier Function
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HUVEC, human umbilical vein endothelial cells (Gibco, Waltham, MA, USA); M200 culture medium (Gibco, Waltham, MA, USA); DMEM High Glucose w/o Sodium Pyruvate w/ L-Glutamine (Euro Clone, Pero, Italy); TNF-α (Sino Biological Inc., Wayne, NJ, USA); Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA); Hyaluronate Lyase from Streptomyces hyalurolyticus (Sigma-Aldrich, St. Louis, MO, USA); Transwell system with filter of 0.4 µm pore size, 6.5 mm diameter (Corning, New York, NY, USA); FITC labelled dextran (Mw~250,000, Sigma-Aldrich, St. Louis, MO, USA); heparinases I–II–III (form F. heparinum, Seikagaku, Tokyo, Japan); 2-Aminoacridone, AMAC-(Sigma-Aldrich, St. Louis, MO, USA); Chondroitinase ABC (from Proteus vulgaris, Seikagaku, Tokyo, Japan); 3-hydroxybiphenol (Fluka, Buchs, Switzerland); D-Glucuronic acid (Sigma-Aldrich, St. Louis, MO, USA); prostaglandin E1 and indomethacin (Sigma); all chemicals were purchased by Sigma-Aldrich (St. Louis, MO, USA).
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