The reaction buffer (RB) consisted of 25 mm Tris-HCl (pH 8.5) and 25 mm NaCl. The β(R)- and β(S)-stereoisomers of GS-HPV were separately generated by incubating racemic β(S)- and β(R)-MPHPV (0.46 mm ) in RB with 5 mm GSH and either 38 μg/ml LigF1 or 36 μg/ml LigE for several hours (Fig. S4 ). This sample, containing a single GS-HPV stereoisomer, guaiacol, and the unreacted MPHPV stereoisomer (as well as LigE or F1), was diluted with RB to achieve the desired concentration of GS-HPV for the time course reaction (0.005, 0.01, 0.02, 0.1, or 0.2 mm ). An additional 5 mm GSH (dissolved in RB) was added before initiation of each time course. At time 0, 100 μl of the indicated enzyme (resuspended in RB) was combined with 1800 μl of the diluted GS-HPV reaction mixture to achieve final concentrations of 8 nm NaGSTNu, 20 nm NaGSTNu (T51A), 100 nm NaGSTNu (Y166F), 10 nm NaGSTNu (Y224F), 195 nm EcYghU, 195 nm EcYfcG, or 47 or 18 nm SYK6GSTNu (for the β(R)- and β(S)-GS-HPV reactions, respectively). Reactions were performed at 25 °C. At specified time points, 300 μl of the reaction was removed and combined with 100 μl of 1 m HCl (Acros Organics, Geel, Belgium) to stop the reaction before HPLC analysis to quantify HPV formed.
1 m hcl
Manufactured by Thermo Fisher Scientific
Sourced in Belgium, United States
1 M HCl is a laboratory reagent that provides a solution of hydrochloric acid with a concentration of 1 mole per liter. It is commonly used in various analytical and experimental procedures in chemistry and biology laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (1 mentions)
Bile salt, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
α amylase, by Merck Group (1 mentions)
Pancreatin, by Merck Group (1 mentions)
5 protocols using 1 m hcl
1
Synthesis and Characterization of GS-HPV Stereoisomers
2
Kinetic Analysis of MPHPV Cleavage
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Various concentrations of racemic (β(S) and β(R)) MPHPV were combined with 5 mm GSH in ERB. At time 0, 100 μl of an indicated enzyme in ERB + 5 mm GSH was combined with 1 ml of the racemic MPHPV/GSH sample (both pre-equilibrated to 25 °C) at 25 °C. The final concentration of β(R)-MPHPV in each reaction was 0.0045, 0.010, 0.017, 0.068, or 0.13 mm . Final enzyme concentrations were 18 nm BaeAB, 23 nm BaeAB (B:S14A), 22 nm BaeAB (A:S15A), 24 nm BaeAB (A:S15A/B:S14A), 98 nm BaeAB (A:N14A), and 70 nm NaLigE (all BaeAB concentrations are for the heterodimers; the NaLigE concentration is for the individual polypeptides that make up the functional homodimers). At different time points, 200 μl of a reaction was removed and combined with 40 μl of 1 m HCl (Acros Organics, Geel, Belgium) to terminate the enzyme reaction before HPLC analysis to quantify GS-HPV formed. Control reactions were allowed to proceed for several hours to ensure that only the β(R)-MPHPV in the reaction mixtures was cleaved.
3
Synthesis of Alg-PBA Conjugate for Biomedical Applications
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The Alg-PBA conjugate was synthesized after conjugating the 3-aminomethylphenylboronic acid (PBA, Alfar Aesar, Ward Hill, MA, USA) to alginate backbone using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methymorpholinium chloride (DMTMM, TCI America, Portland, OR, USA) as the coupling agent following a modified protocol from a previous study [18 (link)]. In general, 100 mg alginate (0.5 mmol, Protanal® LF 10/60, FMC Corporation, Philadelphia, PA, USA) was dissolved in 10 mL de-ionized (DI) water. A total of 23.5 mg PBA (0.125 mmol) and 37.5 mg DMTMM (0.125 mmol), respectively, were added to the alginate solution. After complete dissolution, the pH of the solution was adjusted to 6.5 with 1 M HCl (Fisher Chemical, Hampton, VA, USA) solution. To tune the conjugation ratio, another reaction was conducted using 0.5 mmol alginate, 0.25 mmol PBA, and 0.25 mmol DMTMM at pH 6.5 in 10 mL water. Each reaction mixture was stirred at room temperature for 3 days before being transferred to a 6–8 kDa molecular weight cut-off (MWCO) dialysis bag (Spectrum). The mixture was dialyzed against DI water for 3 days, with the water changed thrice every day at room temperature. The purified conjugate was lyophilized and stored in the freezer before use.
4
Formulation Development of Candurin Tablets
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Candurin was purchased from EMD Performance Materials (Philadelphia, PA). Copovidone (Va64), a water-soluble copolymer of vinylpyrrolidone and vinyl acetate, was purchased from BASF (Ludwigshafen, Germany). Sodium stearyl fumarate was purchased from JRS Pharma LP (Patterson, NY). HPLC grade methanol, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Pittsburgh, PA). Fasted-state simulated intestinal fluid (FaSSIF) was purchased from Biorelevant.com Ltd. (Surrey, United Kingdom). Sodium chloride, potassium salt, and 1 M HCL were purchased from Fisher Scientific. All other reagents used were of ACS grade or higher.
5
Simulated Gastrointestinal Digestion of H. elongata
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The simulation of the gastrointestinal digestion of the H. elongata sample extracts was performed following the method described by Campos et al. [14 (link)]. To simulate the oral digestion, 1 g of dried sample (CRD or EtOAc) was suspended in 20 mL of distilled water, followed by the adjustment of the pH to between 5.6 and 6.9, with 1 M NaHCO3 (Sigma-Aldrich, St. Louis, MO, USA), prior to the addition of 0.6 mL/min of α-amylase (Sigma-Aldrich, St. Louis, MO, USA) at 100 U/mL. Enzymatic digestion was carried out during 2 min of mastication, at 37 °C and 200 rpm. Before moving to the next compartment, the pH of the mouth digest was adjusted to 2.0, using 1 M HCl (Fisher, Pittsburgh, PA, USA), and then mixed with a simulated gastric juice consisting of 25 mg/mL of pepsin (Sigma-Aldrich, St. Louis, MO, USA) added at a ratio of 0.05 mL/mL of mouth digest. Incubation was carried out over 60 min at 37 °C and 130 rpm. Finally, for intestinal digestion the pH of gastric digest was adjusted to 6.0, using 1 M NaHCO3, prior to the addition of a simulated intestinal juice consisting of 2 g/L of pancreatin (Sigma-Aldrich, St. Louis, MO, USA) and 12 g/L bile salts (Sigma-Aldrich, St. Louis, MO, USA) at a ratio of 0.25 mL/mL of gastric digest. The samples were then incubated for 120 min, at 37 °C and 45 rpm, to mimic a long intestinal-digestion process.
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