Oligonucleotides were synthesized using a MerMade 4 (BioAutomation) DNA synthesizer using standard phosphoramidite chemistry. The reagents used for synthesis were from Glen Research. The final dimethoxytrityl (DMT) group was retained for HPLC purification at 90 °C (Agilent PLRP-S column, 250 mm × 4.6 mm; A= 100 mM triethylammonium acetate [TEAA] in 5% [v/v] aqueous acetonitrile [MeCN], B= 100 mM TEAA in MeCN; 5:95 to 30:70 A:B over 30 min at 1 mL/min). Oligonucleotides were subject to detritylation through incubation for 60 min at room temperature in 20% (v/v) aqueous glacial acetic acid. The reaction was quenched by ethanol precipitation. A second round of HPLC was then performed at 90 °C (Agilent PLRP-S column, 250 mm × 4.6 mm; A= 100 mM triethylammonium acetate [TEAA] in 5% [v/v] aqueous acetonitrile [MeCN], B= 100 mM TEAA in MeCN; 0:100 to 25:75 A:B over 40 min at 1 mL/min).
Plrp s column
Manufactured by Agilent Technologies
Sourced in United States
The PLRP-S column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a porous polymer resin with a high surface area, enabling efficient separation and resolution of analytes.
Automatically generated - may contain errors
Lab products found in correlation
1260 infinity, by Agilent Technologies (3 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
1290 series hplc, by Agilent Technologies (2 mentions)
Masshunter bioconfirm software, by Agilent Technologies (2 mentions)
1260 hplc, by Agilent Technologies (2 mentions)
6220 esi tof mass spectrometer, by Agilent Technologies (2 mentions)
Openlab cds software, by Agilent Technologies (2 mentions)
Agilent 1260 hplc system, by Agilent Technologies (2 mentions)
Openlab cds data analysis, by Agilent Technologies (2 mentions)
717 plus autosampler, by Waters Corporation (1 mentions)
Foxy r1 fraction collector, by Teledyne (1 mentions)
Human leptin radioimmunoassay kit, by Merck Group (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Compass dataanalysis v 4.3, by Bruker (1 mentions)
6538 uhd q tof, by Agilent Technologies (1 mentions)
Acquity uplc, by Agilent Technologies (1 mentions)
Masslynx data system, by Waters Corporation (1 mentions)
Microtof q 2 mass spectrometer, by Bruker (1 mentions)
Compass data analysis version 4, by Bruker (1 mentions)
Esi l low concentration tuning mix, by Agilent Technologies (1 mentions)
39 protocols using plrp s column
1
Oligonucleotide Synthesis and Purification
2
Peptide Purification by HPLC
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The peptide was purified in four batches by high performance liquid chromatography (HPLC) on a VWR-Hitachi LaChrom Elite instrument (VWR, Darmstadt, Germany) equipped with a semi-preparative PLRP-S column (325 mm × 30 mm, 30 nm pore diameter, 8 µm particle size; Agilent, Santa Clara, CA, USA), a VWR-Hitachi L2400 UV detector (VWR, Darmstadt, Germany) and a Foxy R1 fraction collector (Teledyne ISCO, Lincoln, NE, USA). A flow rate of 6 mL min -1 and the following gradient (solvent A: water, 0.1% formic acid (FA), solvent B: acetonitrile, 0.1% FA) was applied: 0 min: 5% B, Elite instrument (VWR, Darmstadt, Germany) equipped with a 717 plus autosampler (Waters, Milford, MA, USA), an analytical PLRP-S column (150 mm × 4.6 mm, 30 nm pore diameter, 8 µm particle size; Agilent, Santa Clara, CA, USA), a VWR-Hitachi L2400 UV detector (VWR, Darmstadt, Germany) and an expressionL cms MS device (Advion, Harlow, UK). A flow rate of 1 mL min -1 and the following gradient (solvent A: water, 0.1% FA, solvent B: acetonitrile, 0.1% FA) was applied: 0.0 min: 5% B, 2.5 min: 5% B, 12.5 min: 60% B, 13.5 min: 95% B, 16.0 min: 95% B, 17.0 min: 5% B, 18.5 min: 5% B. A dead time of 2.0 min was observed. TC(4,8) eluted after retention time of t R = 9.8 min. Fractions deemed sufficiently pure were combined and lyophilized. The peptide was obtained as a white fluffy solid (17.8 mg, 8.65 µmol, 5%).
3
Purification and Characterization of Synthetic Aβ40
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Synthetic Aβ40 in powder form
was dissolved in 0.1% NH4OH and assayed for protein concentration
in three ways: a
bicinchoninic acid assay, tyrosine absorbance at 280 nm, and mass
spectrometric determination of Ala, Val, and Leu in an acid hydrolysate.
All three methods showed the material to be 65–70% protein
by weight, with the balance presumably water and/or salts. The purity
of the protein component was determined by two different high-performance
liquid chromatography methods. Method 1 involved a 4.6 mm × 250
mm reversed phase Vydac MS C4 column, mobile phase A composed of 0.1%
NH4OH in water, mobile phase B composed of 0.1% NH4OH in acetonitrile (1/200 in volume), a flow rate of 700 μL/min,
and detection by absorption at 215 nm. The gradient program was 0%
B from 0 to 8 min, with a linear increase to 60% B at 40 min. Method
2 involved a 4.6 mm × 250 mm reversed phase Varian PLRP-S column,
the same mobile phases, a flow rate of 500 μL/min, and detection
by absorption at 278 nm. The gradient program was a linear increase
from 0 to 60% B over 60 min. Only minor peptide impurities were detected
as small peaks eluting earlier than the main peak, barely above the
baseline noise. Method 1 or 2 suggested that the purity of synthetic
Aβ40 in the eluted fraction was >92 or >96%, respectively.
was dissolved in 0.1% NH4OH and assayed for protein concentration
in three ways: a
bicinchoninic acid assay, tyrosine absorbance at 280 nm, and mass
spectrometric determination of Ala, Val, and Leu in an acid hydrolysate.
All three methods showed the material to be 65–70% protein
by weight, with the balance presumably water and/or salts. The purity
of the protein component was determined by two different high-performance
liquid chromatography methods. Method 1 involved a 4.6 mm × 250
mm reversed phase Vydac MS C4 column, mobile phase A composed of 0.1%
NH4OH in water, mobile phase B composed of 0.1% NH4OH in acetonitrile (1/200 in volume), a flow rate of 700 μL/min,
and detection by absorption at 215 nm. The gradient program was 0%
B from 0 to 8 min, with a linear increase to 60% B at 40 min. Method
2 involved a 4.6 mm × 250 mm reversed phase Varian PLRP-S column,
the same mobile phases, a flow rate of 500 μL/min, and detection
by absorption at 278 nm. The gradient program was a linear increase
from 0 to 60% B over 60 min. Only minor peptide impurities were detected
as small peaks eluting earlier than the main peak, barely above the
baseline noise. Method 1 or 2 suggested that the purity of synthetic
Aβ40 in the eluted fraction was >92 or >96%, respectively.
4
Preparative and Analytical HPLC Purification
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Preparative HPLC was performed using a Varian ProStar Model 325 HPLC (Agilent Technologies, Santa Clara, CA) equipped with a fraction collector. Preparative separations utilized a Varian PLRP-S column (150 × 25 mm, 100 Å, 10 µm), with a mobile phase comprising 0.1% (v/v) aqueous NH4OH and acetonitrile (20:80 0.01 min, 45:55 25.00 min, 20:80 26.00 min, stop 30 min) at a flow rate of 25 mL/min.
Analytical HPLC was performed on a C18 analytical column (4.6 mm × 250 mm, 5 μm; Hanbon Sci. & Tech. Huanan, China) coupled with a C18 guard cartridge (4.6 mm × 10 mm, 5 μm; Hedera), maintained at 30 °C. The mobile phase comprised acetonitrile and 0.1 % aqueous TFA, and a gradient method was employed for the analysis (34:66 0.01 min, 41:59 25.00 min, 34:66 25.01 min, stop 30 min). The mobile phase was filtered through a 0.45 μm filter and delivered at a flow rate of 1.0 mL/min. The Ket absorbance was monitored at 254 nm.
Analytical HPLC was performed on a C18 analytical column (4.6 mm × 250 mm, 5 μm; Hanbon Sci. & Tech. Huanan, China) coupled with a C18 guard cartridge (4.6 mm × 10 mm, 5 μm; Hedera), maintained at 30 °C. The mobile phase comprised acetonitrile and 0.1 % aqueous TFA, and a gradient method was employed for the analysis (34:66 0.01 min, 41:59 25.00 min, 34:66 25.01 min, stop 30 min). The mobile phase was filtered through a 0.45 μm filter and delivered at a flow rate of 1.0 mL/min. The Ket absorbance was monitored at 254 nm.
5
Comprehensive Blood Biomarker Analysis
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Blood samples were collected from the antecubital vein between 9:30 and 10:30 am. Peripheral blood tests were performed to determine red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (Hct) levels, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. To quantify the active form of hepcidin, hepcidin-25, serum samples were mixed with synthetic human hepcidin (Peptide Institute, Osaka, Japan) as an internal standard and applied to a reverse-phase PLRP-S column (5 mm, 300 Å, 150 × 3 × 2.1 mm; Varian, Inc, Palo Alto, CA, USA). Levels of hepcidin-25 in the eluate were measured using a 4000 QTRAP liquid chromatography tandem mass spectrometry system (Applied Biosystems, Foster City, CA) (25 (link)). A LABOSPECT 008α automatic colorimetric analyzer (Hitachi High-Technologies Corporation, Schaumburg, IL, USA) was used to measure serum levels of iron. To test levels of iron stored in the body, we measured the concentrations of ferritin in serum using an iatro ferritin kit (LSI Medience Corporation, Tokyo, Japan). The leptin levels were quantified using a human leptin radioimmunoassay kit (EMD Millipore Corporation, Billerica, MA) (26 (link)). The adipokine adiponectin was measured using a human adiponectin latex kit (LSI Medience Corporation) (27 (link)).
6
Reversed-Phase HPLC Purification Protocol
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RP-HPLC was performed using a Varian PLRP-S column (100 Å, 8 μm, 150 × 25 mm). As elution buffer A, 0.1% trifluoroacetic acid in water was used. Elution buffer B was composed of acetonitrile, exclusively. Elution conditions were as followed: 0–3 min 25% buffer B; 3–25 min 25%~50% buffer B; 25–27 min 50%~95% buffer B; 27–28 min 95%~25% buffer B; 28–30 min 25% buffer B at 0.6 mL/min. Samples were previously reduced by incubation with DTT at 37 °C for 30 min before performing RP-HPLC.
7
HPLC Separation Protocols for Diverse Analytes
8
Protein Characterization by LC-MS
9
Rapid LC-MS Analysis of Analytes
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Samples were prepared by adding 25 μL of ultrapure water to 75 μL solutions of analytes in acetonitrile + 0.1% TFA. A PLRP-S column (Agilent) was equilibrated to an 85:15 ratio of solvent A (ultrapure water + 0.1% formic acid) to solvent B (acetonitrile + 0.1% formic acid). Liquid chromatography separations were achieved by linear gradient elution, transitioning from 15% to 95% solvent B over 6 min followed by a 2 min hold at 95% B. The column was re-equilibrated to 15% solvent B for 2 min in between injections of the same sample (two technical replicates run per sample, 2 μL injection volume, 600 μL/min flow rate, 50°C). Two blank runs were implemented between samples to ensure against column holdover of analytes. Electrospray ionization mass spectrometry analysis was carried out in positive mode with a capillary voltage of 2Hz (Agilent 6538 UHD Q-TOF).
10
Characterizing Fc-peptide Products
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Initial analysis of Fc-peptide products was carried out by reversed-phase HPLC/MS of the intact or reduced (5 mM DTT, 55 °C, 30 min) product on the system described below. However, mass accuracy and measurement precision were insufficient to ensure unambiguous determination of the state of the peptide carboxy terminus. Consequently, samples were digested with HRV3C protease (PreScission, GE Healthcare Bio-Sciences, Pittsburgh, PA). Digestions of Fc-peptide were performed for 5 h at 5 °C in a buffer consisting of 20 mM TrisHCl, pH 7.0, 150 mM NaCl, 1 mM DTT using one unit of enzyme for up to 100 μg of protein. Polypeptides resulting from the digestion were chromatographically resolved on a Waters (Milford, MA) Acquity UPLC with an Agilent (Santa Clara, CA) PLRP-S column (2.1 × 50 mm, 5 μ dp, 1000 Å) using a multi-linear gradient. Buffer A was 0.1 % formic acid, and buffer B was 0.1 % formic acid in acetonitrile. The peptides were mass analyzed on a Waters API US mass spectrometer scanned from m/z 500–3000 at a rate of 1 Hz. The peptide m/z data was deconvoluted to the single-charge mass domain by the MaxEnt3 algorithm implemented in the Waters MassLynx data system.
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