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High-Performance Liquid Chromatography
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Joel J. Kirschbaum, Adorjan Aszalos
Active fractions were separated using a diol column equilibrated with 0.1 M sodium acetate, pH 7.5 (0.01% thiodiglycol)-n-propanol (20:80) flowing at 1.3 ml/min. Elution was with a linear gradient of n-propanol changing from 72.5 to 50% in 4 hr, at a flow rate of 0.25 ml/min [132]. At least three peaks were found after diverting 1% to a fluorescence detection system [133] involving derivatization with fluorescamine.
Amino Acid Analysis
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
The use of fluorogenic reagents to detect amino acids has been increasing during the past decade. Fluorescamine (4-phenylspiro[furan-2-(3H), 1’-phthalan]-3,3’-dione) was introduced by Udenfriend and co-workers50 for the detection of amino acids. At the time, the reagent provided greater sensitivity for most amino acids than did the ninhydrin reagent but did not detect amino acids such as proline or hydroxyproline. Felix and Turkelsen51 reported the use of post-column derivatization of proline with N-chlorosuccinimide which permitted effective reaction with fluorescamine. Further studies52 on the chemistry of the reaction of fluorescamine with amino acids have created a solid base for the use of this reagent for amino acid analysis using colorimetric rather than fluorescence detection. Further developments of note in the use of this reagent include the use of alkaline sodium hypochlorite for the post-column derivatization of amino acids. A recent review of this technique has appeared.22
Spectroscopy and Fluorimetry
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
The more popular method of producing a fluorescent species has been the chemical coupling or condensation method (Table 4.8). This has been a productive approach in the assay of amines especially using fluorescamine (Figure 4.23). A commercial version of the reagent is available (Fluram) as described for the analysis of the biogenic amines tryptophan, noradrenaline, dopamine, and the drug, daunorubicin.380
Colistin conditioning surfaces combined with antimicrobial treatment to prevent ventilator-associated infections
Published in Biofouling, 2022
Diana Alves, Hélder Lopes, Idalina Machado, Maria Olívia Pereira
The amount of COL physically adsorbed onto adhesion surfaces was estimated by quantifying the amount of unattached antibiotic in the solution retrieved immediately after completing the conditioning process. Colistin concentration was measured using a fluorescamine (Sigma) assay (Ko et al. 2013). Fluorescamine assay was performed by mixing fluorescamine solution (3 mg ml−1 in acetone) and the sample at 1:3 ratio in a 96-black-well plate (Greiner). After 15 min of incubation at room temperature, the fluorescence intensity of each sample was measured by using a microplate reader (Synergy HT, Biotek). Adsorption efficiency was represented as the percentage ratio of the amount of adsorbed COL to the initial amount of COL. To evaluate the possible release of physically adsorbed COL to the adhesion surfaces, COL-conditioned wells were filled with UP water and kept in the same conditions used for biofilm formation (at 37 °C, 120 rpm, for 24 h). After this period, COL released was also measured by the fluorescamine assay. Experiments were performed in triplicate.
Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3
Published in OncoImmunology, 2020
Mathilde Boulpicante, Romain Darrigrand, Alison Pierson, Valérie Salgues, Marine Rouillon, Benoit Gaudineau, Mehdi Khaled, Angela Cattaneo, Angela Bachi, Paolo Cascio, Sébastien Apcher
Reconstitution of REG-20S complexes and the degradation of short fluorogenic substrates, the MP-45 and the KH-52 polypeptides (containing the SIINFEKL epitope in their middle) or the MP-46 and the KH-53 polypeptides (containing the MBP epitope in their middle), were performed according to previously described methods.35–37 Briefly, PSME3- and REGα/β-20S proteasomes were reconstituted by preincubating human 20S constitutive or immuno particles (BostonBiochem, USA) with a 6-fold molar excess of PSME3 (BostonBiochem, USA) or REGαβ36 at 37°C for 30 min in 20 mM HEPES, pH 7.6, and 2 mM NaCl and were immediately used for degradation experiments. For kinetic analysis and to generate peptide products for MS/MS studies, all polypeptides (50 µM) were incubated with 20S, PSME3-20S or REGα/β-20S (20 nM) for 8 h at 37°C in 20 mM HEPES, pH 7.6, 2 mM NaCl. To assay the peptides generated during protein degradation, we measured the appearance of new amino groups using fluorescamine as previously described.37 In brief, at the end of the incubation, the peptide products were separated from undegraded polypeptides by ultrafiltration through a membrane with a 3-kDa cutoff (Nanosep, Pall, USA), and these samples were assessed with fluorescamine (Sigma) and used for MS/MS analysis.
Sustained release and pharmacologic evaluation of human glucagon-like peptide-1 and liraglutide from polymeric microparticles
Published in Journal of Microencapsulation, 2019
Luis Peña Icart, Fernando Gomes de Souza, Luís Maurício T. R. Lima
Polyvinyl alcohol Mw 89 kDa-98kDa, >99% hydrolysed (PVA; lot # MKBD2262V, Cat: 9002-89-5) was obtained from Sigma-Aldrich. Liraglutide (Victoza®, Lots # FS60K24 and FS6W992) was purchased at local drug store, and GLP1 (7–37, sequence ‘HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG,’ >95% purity, lot #98664) was purchased from Genemed Synthesis Inc (USA). Fluorescamine was obtained from Sigma-Aldrich (USA; Cat #F9015). The branched PLGA-glucose (lots 0811001762 and Cat #5004-A) (Bodmer et al.1992) and the linear PLGA 50:50 Lactide/Glycolide (lot 14007) were purchased from Purasorb. Linear PLGA 85:15 (Cat. #430471), 75:25 (Cat. #P1941) and 65:35 (Cat. #P2066) Lactide/Glycolide (lots #MK861113V, #089K134V and #050M1668V, respectively) were purchased from Sigma-Aldrich. PLA, PLA-PEG and PLA-PEG-F (fluorescein) were synthesised and characterised as described elsewhere (Icart et al.2016). All other reagents were from analytical grade. All reactants were used as received.