Five mg of lyophilized PEP04314 (0.71 µmol, Affibody AB, Solna, Sweden) was added to 0.5 mL of 0.2 M ammonium acetate buffer (pH 7.5), and the solution was added to a reaction vial containing 0.4 mg (0.72 µmol) of (±)-H3RESCA-maleimide or maleimide-mono-amide-NOTA (Macrocyclics, Plano, Tx, USA). The reaction mixture was kept at room temperature for 90 min before being transferred to an ultracel 3K centrifugal filter (Merck) containing 3 mL of 0.1 M ammonium acetate buffer (pH 4) and centrifuged at 4000 rpm for 90 min. The flow-through was discarded, and 4 mL of fresh 0.1 M ammonium acetate buffer (pH 4) was added. The filter was then centrifuged again for 90 min, and the flow-through was discarded. Purified (±)-H3RESCA-PEP04314 or H2NOTA-PEP04314 was collected via reverse spin in 1 mL of 0.1 M ammonium acetate buffer (pH 4.5), and stored at -70 °C in 100 µL aliquots prior to use. Purity of the final product was determined via a Waters Acquity LC/MS system equipped with a Waters Xselect CSH C18 column (250 mm ×10 mm, 130 Å) at a flow rate of 5 mL/min using a gradient of EtOH (10-40% over 15 min) and 0.1% formic acid.
Xselect csh c18 column
Manufactured by Waters Corporation
Sourced in United States, Ireland
The XSelect CSH C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It features a C18 stationary phase and is designed for the separation and analysis of a wide range of organic compounds.
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Lab products found in correlation
Vanguard cartridge, by Waters Corporation (3 mentions)
Anp stock, by New England Biolabs (2 mentions)
Q exactive ms, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Xbridge amide column, by Waters Corporation (2 mentions)
Acquity lcms system, by Waters Corporation (1 mentions)
Ultracel 3k centrifugal filter, by Merck Group (1 mentions)
2475 fluorescence detector, by Waters Corporation (1 mentions)
1525 binary pump, by Waters Corporation (1 mentions)
2998 photodiode array detector, by Waters Corporation (1 mentions)
Avance 3 400 mhz spectrometer, by Bruker (1 mentions)
Avance 3 500 mhz spectrometer, by Bruker (1 mentions)
Quaternary solvent manager r, by Waters Corporation (1 mentions)
Fluoromax 4 spectrofluorometer, by Horiba (1 mentions)
1220 infinity compacted lc series, by Agilent Technologies (1 mentions)
Delta 600 hplc, by Waters Corporation (1 mentions)
Alliance hplc system model 2695, by Waters Corporation (1 mentions)
Quattro lc micromass, by Waters Corporation (1 mentions)
Xselect csh c18, by Waters Corporation (1 mentions)
1260 system, by Waters Corporation (1 mentions)
27 protocols using xselect csh c18 column
1
Radiolabeling of Affibody Molecules
2
HPLC Analysis of Purified CST
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HPLC analysis was performed as previously described [20 (link)]. Briefly, samples were run on a XSelect CSH C18 column (3.5 μm, 2.1 × 150 mm; Waters) using a mobile phase of Buffer B (0.1 M K•PO4 pH 6.5, 8 mM tetrabutylammonium hydrogensulfate (TBHS)). For samples containing known compounds, stocks of the compound were diluted into Buffer B. For samples containing protein extracts, a volume of typically 50 μM purified CST in Buffer A was added to an equal volume of a phenol-chloroform-isoamyl alcohol mix (25:24:1) in a 1.5 ml microcentrifuge tube. This mixture was vigorously vortexed for 1 min and then spun at 21,000 x g for 5 min in a microcentrifuge. The top aqueous layer was collected and added to an equal volume of chloroform. This was again vortexed for 1 min and then spun at 21,000 x g for 5 min in a microcentrifuge. The top aqueous layer was collected for HPLC analysis. For AnP-treatment of CST prior to phenol-chloroform extraction, 3 μl AnP stock (5,000 units/ml, New England Biolabs) was added to 50 μM purified CST in Buffer A along with 0.5 mM ZnCl2 and 1mM MgCl2. For protein samples that were dialyzed prior to phenol-chloroform extraction, 100 μl aliquots of 50 μM purified CST were placed in a dialysis cassette with a 10K MWCO and dialyzed against 100 ml at 4°C with at least three separate buffer exchanges over the course of 24 hr.
3
CLOP Quantification via RP-HPLC
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Analysis of CLOP samples took place using reversed-phase Agilent 1200 Infinity Quaternary System HPLC using an Xselect® CSH™ C18 column with a 3.5-µm particle size and a 3.0 × 150-mm dimension (Waters Corporation, MA, USA). The best peak shape was achieved using a mobile phase consisting of 70% acetonitrile and 30% water (pH 2.5 ± 0.05) at a UV absorbance of 230 nm. The injection volume was set at 10 µL, and the flow rate was set at 0.5 mL/min. A standard calibration curve was plotted within a concentration range of 0.1–50 µg/mL. This curve was then applied to each of the release samples in order to quantify the amount of CLOP present in each sample.
4
Peptide Fractionation and Pooling
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The dried peptide mixture was dissolved in 55 μL of mobile phase A (10 mM ammonium hydroxide). 50 μL of the sample was injected onto a 2.1 × 150 mm XSelect CSH C18 column (Waters) equilibrated with 3% mobile phase B (10 mM ammonium hydroxide, 90% Acetonotrile). Peptides were separated using a similar gradient as previously described14 (link) with the following gradient parameters (Table S2 -Supplemental File 1 ) at a flow rate of 0.2 mL/min. 60 peptide fractions were collected corresponding to 2.5 min each. Ten pooled samples were generated by concatenation15 (link) in which every 10th fraction was combined (ie: 1, 11, 21, 31, 41, 51; next 2, 12, 22, 32, 42, 52, etc., six pools total).
5
Analytical Characterization of Organic Compounds
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1H and 13C{1H} NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer or a Bruker AVANCE III 500 MHz spectrometer in CDCl3. Spectra were referenced internally by using the residual solvent (1H: δ 7.26) or solvent (13C: δ 77.2) resonance relative to SiMe4. ESI mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. Electronic absorption and steady‐state fluorescence spectra were taken on a Cary 5G UV–Vis–NIR spectrophotometer and a HORIBA FluoroMax‐4 spectrofluorometer, respectively. Reverse‐phase HPLC separation was performed on an Apollo‐C18 column (5 µm, 4.6 mm × 150 mm) at a flow rate of 1 mL min−1 for analytical purpose or on a XBridge BEH300 Prep C18 column (5 µm, 10 mm × 250 mm) at a flow rate of 3 mL min−1 for preparative purpose, using a Waters system equipped with a Waters 1525 binary pump and a Waters 2998 photodiode array detector. The solvents used for HPLC analysis were of HPLC grade. LC‐MS studies were performed on a XSelect CSH C18 column (5 µm, 4.6 mm × 250 mm) at a flow rate of 0.8 mL min−1 using a Waters system equipped with a Waters Quaternary Solvent Manager‐R, a Waters 2998 photodiode array detector, a Waters 2475 fluorescence detector, and a Waters single quadrupole detector 2. The solvents used were of LC‐MS grade.
6
Simultaneous HPLC Analysis of Curcumin and DPA
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Simultaneous analysis of CUR and DPA was conducted by utilising an HPLC system consisting of an Agilent Technologies 1220 Infinity compacted LC series (Agilent Technologies UK Ltd., Stockport, UK) with a degasser, binary pump, standard autoinjector and UV detector. All analyte separation was carried out on an XSelect CSH C18 column (Waters, 3.0 × 150 mm) with a particle size of 3.5 μm and a pore size of 130 Å, fitted with a VanGuard® cartridge (Waters, 3.9 × 5 mm). The mobile phase consisted of a mixture of 0.001% v/v of phosphoric acid in water (mobile phase A) and acetonitrile (ACN) (mobile phase B). The analysis was performed at room temperature for 15 min using the gradient condition, as detailed in Table 1 . The injection volume of all samples was 20 µL, and the flow rate was 0.8 mL min−1.
7
HPLC Analysis for Compound Purity Monitoring
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Example 9
The method monitoring the reaction of example from 1 to 8 and the purity of the compound of formula (I) and formula (II), via HPLC:
-
- Column: Waters XSelect CSH C18 Column, 130 Å, 3.5 μm, 4.6 mm×150 mm;
- Temp. Column: 40° C.;
- Mobile Phase A: Phosphate buffer (Transfer 1.31 g of KH2PO4, accurately weighed, in 1000 mL volumetric flask, add 500 mL of Water milliQ and well mix. Add 26.7 μL of H3PO4 85%, accurately measured, and bring to volume; Filter the solution trough a 0.22 μm Millipore filter then degas; Check the pH; the pH should be approximately 3.65±0.05, If the pH is not between 3.60 and 3.70, adjust the pH with 85% Phosphoric Acid until it is between 3.60 and 3.70);
- Mobile Phase B: Acetonitrile;
- Gradient
-
- Flow: 1.0 mL/min;
- UV Detector: 260 nm;
- Injection Volume: 5 μL;
- Analysis Time: 35 min;
- Diluent: H3PO4 0.1%/Methanol 1/9.
8
Enzymatic Synthesis of 2/3-O-AADPr
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A one-step enzymatic reaction was
used to convert NAD+ into 2/3-O-AADPr.
The residue Glu179 from NAD-glycosylhydrolase of Aplysia californica is crucial for catalysis and the E179G
mutation prevents NAD+ hydrolysis.45 (link) NAD+ (50 mM) was added to 1 M sodium acetate at pH 5.5.
Mutant E179G NAD+-glycosylhydrolase (25 μM) was added
to the solution and reacted overnight at 25 °C to quantitatively
provide 1-α-OAADPr that rapidly and fully isomerizes to 2/3-O-AADPr. The 2/3-O-AADPr species were purified
and resolved via HPLC using a 0–30% gradient of H2O to acetonitrile (with 0.05% TFA) on a Waters Delta 600 HPLC and
a Waters XSELECT CSH C18 column (5 μm; 4.6 × 250 mm). Identity
of the compounds was confirmed by mass spectrometry with an exact
mass of 600.0793 m/z [M–H]
and by 1H and 13C NMR that matched the previously
reported compound (SI Figure S1 ).46 (link)
used to convert NAD+ into 2/3-O-AADPr.
The residue Glu179 from NAD-glycosylhydrolase of Aplysia californica is crucial for catalysis and the E179G
mutation prevents NAD+ hydrolysis.45 (link) NAD+ (50 mM) was added to 1 M sodium acetate at pH 5.5.
Mutant E179G NAD+-glycosylhydrolase (25 μM) was added
to the solution and reacted overnight at 25 °C to quantitatively
provide 1-α-OAADPr that rapidly and fully isomerizes to 2/3-O-AADPr. The 2/3-O-AADPr species were purified
and resolved via HPLC using a 0–30% gradient of H2O to acetonitrile (with 0.05% TFA) on a Waters Delta 600 HPLC and
a Waters XSELECT CSH C18 column (5 μm; 4.6 × 250 mm). Identity
of the compounds was confirmed by mass spectrometry with an exact
mass of 600.0793 m/z [M–H]
and by 1H and 13C NMR that matched the previously
reported compound (
9
Quantitative Cecal Bile Acid Analysis
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Cecal bile acid levels were identified and quantified by high‐pressure liquid chromatography‐electrospray‐mass spectrometry/mass spectrometry (HPLC‐ES‐MS/MS) by recent published method suitable for use in pure standard solution, intestinal content, and stool samples after appropriate pre‐analytical procedures. Liquid chromatography analysis was performed using an Alliance HPLC system model 2695 from Waters combined with a triple quadruple mass spectrometer QUATTRO‐LC (Micromass; Waters) using an electrospray interface. The analytical column was a Waters XSelect CSH C18 column, 5 µm, 150 × 2.1 mm, protected by a self‐guard column Waters XSelect CSH C18 5 µm, 10 × 2.1 mm. BAs were separated by elution gradient mode with a mobile phase composed of a mixture ammonium acetate buffer 15 mM, pH 8.0 (Solvent A) and methanol (Solvent B). Chromatograms were acquired using the mass spectrometer in multiple reaction monitoring mode. Briefly, aliquots of cecal sample homogenate (0.3 g) were extracted with 0.9 ml of isopropanol. The mixture was stirred for 30 min at 37°C, then centrifuged at 800 g for 5 min. The supernatant was then diluted 1:10 (v/v) with 40% isopropanol in 15 mM ammonium acetate at pH 8.00, filtered, transferred to an autosampler vial, and 5 μl injected into the HPLC‐ESI‐MS system.
10
Isoflavone Quantification via HPLC
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The isoflavone concentration was analyzed by high-performance liquid chromatography (HPLC) (Ma et al., 2015 (link)). Briefly, 0.1 g of seed powder was extracted with 5 ml of extraction solution at room temperature overnight. After centrifugation, the supernatant was subjected to HPLC on an Agilent 1260 system equipped with a Waters XSelect CSH C18 column (4.6 × 75 mm in length, 2.5 μm × 75 mm, 2.5 μm). By comparing the retention time and the maximum UV absorbance for the three standards with our samples, we accurately determined the levels of isoflavone components based on the UV absorption value at 260 nm.
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