All solvents and reagents were purchased from commercial sources and used as received unless otherwise stated. 1H- and 13C-NMR spectra were recorded on a Varian VNMRS direct drive Varian console spectrometer operating at 400 and 100 MHz, respectively. Liquid chromatography coupled to mass spectrometry (LCMS) for ligands and complexes were performed using a Waters analytical HPLC system connected to a Waters QtofMS-XEVO (ESI, positive mode) mass spectrometer. Cyclic voltammograms were obtained in a nitrogen atmosphere at 22 °C using a BASi EC Epsilon potentiostat equipped with a 1.6 mm gold working electrode, a platinum wire auxiliary electrode, and quasi silver/silver chloride reference electrode. Measurements were performed in water (molecular biology reagent grade, Sigma) with KCl (1 M) as the supporting electrolyte.
Analytical hplc system
Manufactured by Waters Corporation
The Analytical HPLC system from Waters Corporation is a high-performance liquid chromatography instrument designed for the separation, identification, and quantification of complex mixtures. The system includes a solvent delivery unit, an autosampler, a column compartment, and a detector to provide accurate and reliable results for a wide range of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ec epsilon potentiostat, by Bioanalytical Systems (1 mentions)
Molecular biology reagent grade, by Merck Group (1 mentions)
Prism v5, by GraphPad (1 mentions)
Rp c18 column, by Waters Corporation (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Human male ab plasma, by Merck Group (1 mentions)
9 protocols using analytical hplc system
1
Characterization of Organometallic Complexes
2
Peptide digestion and HPLC analysis
3
Quantification of Entomopathogenic Beauveria Compounds
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The 2 × 106E. rugulosa spores were grown in 50 ml Arg medium for 10 d. Only Arg medium was used for comparative ECB production and transcriptional studied of ecdB knockout background. ECB production was initially tested by confrontation assay (susceptibility) against C. albicans followed its detection by HPLC. Briefly, confrontation assay was done by placing filter disc containing extract on pre-inoculated C. albicans cells in YEPD agar plates. Zone of inhibition formed by E. rugulosa extract against C. albicans was measured. Samples for HPLC analysis were performed as method opted by Hu et al., with minor modifications [26 (link)]. The harvested culture was lyophilized followed by methanol extraction at 30 °C for overnight. The filtered sample was run on analytical HPLC system (Waters) using an RP-C18 column (4.5 × 250 mm, I.D.WAT054375) using 20 μl sample injection. The standard of ECB (Santa Cruz Biotechnology: SC-362020) was run and monitored at 222 nm. The ECB concentration was calculated by peak(s) area and statistical analysis was applied using GraphPad Prism v5.01 (GraphPad Software, Inc.).
4
Enzymatic Digestion and HPLC Analysis
5
Cyclic γ-AA peptides Proteolysis
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Cyclic γ-AApeptides M-3-6-D (0.1 mg/mL) were incubated with 0.1 mg/mL protease in 100 mmol/L ammonium bicarbonate buffer (pH 7.8) at 37 °C for 24 h. Then, the reaction mixtures were concentrated in a speed vacuum to remove water and ammonium bicarbonate. The resulting residues were re-dissolved in H2O/MeCN and analyzed on a Waters analytical HPLC system.
6
Proteolytic Digestion of p53 Proteins
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Lead compounds and peptide
control p53 (0.1 mg/mL) were incubated with 0.1 mg/mL proteases in
100 mM ammonium bicarbonate buffer (pH 7.8) at 37 °C for 24 h.
Then, the reaction mixtures were concentrated in a speed vacuum at
medium temperature to remove water and ammonium bicarbonate. The resulting
residues were re-dissolved in H2O/MeCN and analyzed on
a Waters analytical HPLC system with 0.8 mL/min flow rate and 5–100%
linear gradient of solvent B (0.1% TFA in acetonitrile) in A (0.1%
TFA in water) over the duration of 50 min. The UV detector was set
to 215 nm.
control p53 (0.1 mg/mL) were incubated with 0.1 mg/mL proteases in
100 mM ammonium bicarbonate buffer (pH 7.8) at 37 °C for 24 h.
Then, the reaction mixtures were concentrated in a speed vacuum at
medium temperature to remove water and ammonium bicarbonate. The resulting
residues were re-dissolved in H2O/MeCN and analyzed on
a Waters analytical HPLC system with 0.8 mL/min flow rate and 5–100%
linear gradient of solvent B (0.1% TFA in acetonitrile) in A (0.1%
TFA in water) over the duration of 50 min. The UV detector was set
to 215 nm.
7
Intracellular Ascorbate and Glutathione Analysis
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The intracellular D-erythroascorbic acid content was measured by employing known techniques [28 (link), 29 (link)]. Samples were applied to an analytical HPLC system (Waters) that was equipped with a Waters 460 electrochemical detector. The resulting extracts were separated with ZORBAX SB-C18 columns (Agilent, 250 mm × 4.6 mm) and eluted with 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min.
Cellular GSH was labeled with monobromobimane (mBBr) and measured as described previously [30 (link)]. The mBBr-derivatized thiol compounds were analyzed by HPLC with a Hewlett-Packard 1050 series fluorescence detector with ZORBAX SB-C18 columns (Agilent, 250 mm × 4.6 mm). Samples were eluted with 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min. The mobile phase consisted of 15% methanol and 85% trifluoroacetic acid (0.1%) at a wavelength at 370 nm.
Cellular GSH was labeled with monobromobimane (mBBr) and measured as described previously [30 (link)]. The mBBr-derivatized thiol compounds were analyzed by HPLC with a Hewlett-Packard 1050 series fluorescence detector with ZORBAX SB-C18 columns (Agilent, 250 mm × 4.6 mm). Samples were eluted with 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min. The mobile phase consisted of 15% methanol and 85% trifluoroacetic acid (0.1%) at a wavelength at 370 nm.
8
Hyperoxidized Prx1 Binding Assays
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Srx activity and fluorescence polarization binding assays with hyperoxidized Prx variants were performed as previously described [36 (link)]. Briefly, 50 μM hyperoxidized Prx1, 10 μM WT Srx or variant, 50 mM Tris pH 7.5, 100 mM KCl, 1 mM ATP, 1 mM MgCl2, and 2 mM DTT were incubated in a 30 µL reaction and stopped at various times by the addition of 15 μL 1 M H3PO4. Five µL of the sample was injected onto a Waters analytical HPLC system. Different species were separated on a C4 column (Vydac) using a 60–63% acetonitrile/0.1% TFA gradient over 19 min. The fraction of hyperoxidized Prx1 is reported as the mean ± S.D based on peak areas. For the binding studies MgCl2 was omitted to prevent turnover, and the binding curves were fit to a single site, saturable model using SigmaPlot.
9
Serum Stability Evaluation of Peptide P6
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