Avicularia spec. (“Amazonas Purple”, Peru) venom was fractionated via reversed-phase high-performance liquid chromatography (RP-HPLC) on a Prominence HPLC system (Shimadzu Scientific Instruments, Rydalmere, NSW, Australia). Venom was loaded onto a C18 RP-HPLC column (Phenomenex Jupiter; 250 Å, 250 × 4.6 mm, 5 μm), and fractionated using the following gradient: 5% solvent B (0.043% trifluoroacetic acid (TFA) in 90% acetonitrile) in solvent A (0.05% TFA in H2O) for 5 min, 5–20% solvent B over 5 min, 20–40% solvent B over 40 min, then 40–80% solvent B for 5 min (flow rate 1 mL/min). The peak containing the active peptide (termed ω-Avsp1a, see Section 3.1 for details) was further purified by Hydrophilic Interaction Liquid Chromatography (HILIC) using a Grace VisionHT HILIC column (150 × 4.6 mm, 5 μm) on a Shimadzu Prominence HPLC system with a flow rate of 1 mL/min and using the same solvents as for the initial RP-HPLC run. After 3 min at 95% solvent B, a linear gradient of 95–75% B was run over 20 min, followed by a decrease from 75 to 5% B within 2 min.
Prominence hplc system
Manufactured by Shimadzu
Sourced in Japan, United States, Australia, France, United Kingdom, Germany
The Prominence HPLC system is a high-performance liquid chromatography instrument manufactured by Shimadzu. It is designed for the separation, identification, and quantification of chemical compounds in a sample. The system includes a solvent delivery unit, an autosampler, a column oven, and a variety of detection modules to meet the specific needs of analytical applications.
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Lab products found in correlation
Spd m20a, by Shimadzu (8 mentions)
Sil 20ac, by Shimadzu (7 mentions)
Labsolutions software, by Shimadzu (6 mentions)
Lc 20ad pump, by Shimadzu (6 mentions)
Spd 20a, by Shimadzu (6 mentions)
Lc 20ad, by Shimadzu (6 mentions)
Hemolysate reagent, by Helena Laboratories (4 mentions)
Luna c18, by Phenomenex (4 mentions)
Spd 20a 20av series prominence hplc uv vis detectors, by Shimadzu (4 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (4 mentions)
Waters 1525 binary hplc pump, by Waters Corporation (4 mentions)
Waters 996 photodiode array detector, by Waters Corporation (4 mentions)
Dgu 20a5, by Shimadzu (4 mentions)
4000 qtrap mass spectrometer, by AB Sciex (3 mentions)
Rid 20a, by Shimadzu (3 mentions)
Lc solution software, by Shimadzu (3 mentions)
Rp 18 f254s plates, by Merck Group (3 mentions)
Inertsil ods 3 column, by GL Sciences (3 mentions)
C18 column, by Shimadzu (3 mentions)
Rp c18 silica gel, by Merck Group (3 mentions)
232 protocols using prominence hplc system
1
Purification of Amazonas Purple Venom
2
Glycopeptide Separation and Analysis by HPLC-MS/MS
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Glycopeptide mixtures were separated by the Prominence HPLC system (Shimazu, Kyoto, Japan) using a PEGASIL ODS 3 μm 1 mm i.d. × 100 mm column, and by the mobile phase of 0.1% formic acid (FA) and acetonitrile containing 0.1% FA with linear gradient mode at room temperature. The flow rate was 50 μL/min, and the detection was performed on an Esquire 3000 plus mass spectrometer equipped with an electrospray ionization interface (Bruker Daltonics GmbH, Bremen, Germany). The mass spectrometric parameters were as follows: polarity = positive ion mode, ion source gas (nitrogen) pressure = 10 psi, dry gas = 4.0 L/min, dry temperature = 250 °C and scan range = from 400 to 2000 m/z. In MS/MS experiments, the precursor isolation window was set to ±2 Da, and He was used as the collision gas. The operation was carried out on trapControl software and data were acquired and processed with Compass DataAnalysis (Bruker Daltonics GmbH, Bremen, Germany).
3
Platinum(IV) Anticancer Compounds Characterization
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Materials All reagents were purchased from commercial sources and used without further purification. Solvents were used as received, except for THF, which was dried using an MBraun SPS-800 solvent purification system. Ethacrynic acid (EA) was purchased from Abcam Singapore and GST (from human placenta, 25-125 units/mg protein) was purchased from Sigma-Aldrich. Cisplatin (cDDP), oxoplatin [cis-,cis-,transdiamminedichlorodihidroxo platinum (IV)], and 1 were synthesized according to literature methods.
General Instrumentation 1 H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in parts per million relative to residual solvent peaks. Electrospray ionization mass spectra (ESI-MS) were obtained using a Thermo Finnigan MAT ESI-MS system in negative ion mode. UV-Vis readings for the enzyme assays were obtained on a BioTek Synergy H1 hybrid microplate reader. Pt levels were determined on a Perkin-Elmer Optima ICP-OES spectrometer by CMMAC, NUS. The purity of Pt(IV) compounds were determined using analytical HPLC on a Shimadzu Prominence HPLC system, with a Shimpack VP-ODS C18 (5 μM, 120 Å, 150 mm × 4.60 mm i.d) column at r.t. at a flow rate of 1.0 mL/min with UV detection at 254 nm and 280 nm. Reduction studies were carried out on the same system with modified conditions (described below).
General Instrumentation 1 H NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in parts per million relative to residual solvent peaks. Electrospray ionization mass spectra (ESI-MS) were obtained using a Thermo Finnigan MAT ESI-MS system in negative ion mode. UV-Vis readings for the enzyme assays were obtained on a BioTek Synergy H1 hybrid microplate reader. Pt levels were determined on a Perkin-Elmer Optima ICP-OES spectrometer by CMMAC, NUS. The purity of Pt(IV) compounds were determined using analytical HPLC on a Shimadzu Prominence HPLC system, with a Shimpack VP-ODS C18 (5 μM, 120 Å, 150 mm × 4.60 mm i.d) column at r.t. at a flow rate of 1.0 mL/min with UV detection at 254 nm and 280 nm. Reduction studies were carried out on the same system with modified conditions (described below).
4
Alfaxalone Pharmacokinetics in Anesthesia
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Blood samples of 10 ml were collected from the right external jugular
catheter at 15, 30, 45 and 60 min of anesthesia and immediately after standing in group
AGM. All blood samples were immediately placed on ice, and the plasma was separated from
blood and frozen at −20°C.
Alfaxalone was recovered from plasma samples by liquid-liquid extraction using methyl
tert-butyl ether, with 11-hydroxy progesterone as the internal standard. The extracted
substance was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by
electrospray ionization in positive ion mode. LC-MS/MS system consisted of Shimadzu
prominence HPLC system (Shimadzu Co., Tokyo, Japan) and AB Sciex QTRAP 4000 mass
spectrometer (AB Sciex, Framingham, MA, U.S.A.). The calibration was performed using
linear standard curves in the range of 10–500 mg/l, and the coefficient
of correlation (r2) exceeded 0.995. The lower limit of quantification was 10
ng/ml, and the recovery and repeatability were 94.0
and 4.8%, respectively. Analysis of data was performed using SAAM-II program (University
of Washington, Seattle, WA, U.S.A.).
catheter at 15, 30, 45 and 60 min of anesthesia and immediately after standing in group
AGM. All blood samples were immediately placed on ice, and the plasma was separated from
blood and frozen at −20°C.
Alfaxalone was recovered from plasma samples by liquid-liquid extraction using methyl
tert-butyl ether, with 11-hydroxy progesterone as the internal standard. The extracted
substance was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by
electrospray ionization in positive ion mode. LC-MS/MS system consisted of Shimadzu
prominence HPLC system (Shimadzu Co., Tokyo, Japan) and AB Sciex QTRAP 4000 mass
spectrometer (AB Sciex, Framingham, MA, U.S.A.). The calibration was performed using
linear standard curves in the range of 10–500 mg/l, and the coefficient
of correlation (r2) exceeded 0.995. The lower limit of quantification was 10
ng/ml, and the recovery and repeatability were 94.0
and 4.8%, respectively. Analysis of data was performed using SAAM-II program (University
of Washington, Seattle, WA, U.S.A.).
5
HILIC-MS Analysis of Biomolecules
6
Quantitative HPLC Analysis of OVA
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High performance liquid chromatography (HPLC) analysis was performed with a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) coupled with a SPD-M20A Photodiode Array Detector (Shimadzu Corp., Kyoto, Japan). Chromatographic separation was performed using Jupiter C4 column (5 μm, 300 Å, 150 × 4.6 mm id, Phenomenex) proceeded by a 20 × 4.6 mm guard column. The column oven was maintained at 40°C and the auto-sampler temperature at 4°C. Mobile phase A (water, 0.025% TFA) and mobile phase B (acetonitrile, 0.025% TFA) was used as solvent system. Gradient elution of mobile phase B was initiated from 15% (1 min), increased from 15% to 100% (8 min), and then held at 100% for 11 min, total flow rate was 1.0 mL/min. Ultraviolet (UV) absorption spectra were monitored at 214 nm. Experimental scheme of OVA tor HPLC analysis is Fig 1 .
7
Serum Metabolomics Protocol via Orbitrap
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The serum untargeted metabolomics data were quantified through a Shimadzu Prominence HPLC system (Shimadzu) coupled to an LTQ Orbitrap Velos instrument (Thermo Fisher Scientific) at the CAS Key Laboratory of Separation Science for Analytical Chemistry. The details of analytical conditions were as described in a previous study61 (link). The quantitation of bile acid profiles was performed by Metabo-Profile62 (link)–64 (link).
8
Comprehensive Lipidomic Analysis by HPLC-MS
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Semi-quantitative lipidomics was performed with a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan) and an LTQ Orbitrap mass spectrometer (Thermo-Fisher Scientific Inc., San Jose, CA) using electrospray ionization (ESI) mode in both positive and negative modes. The HPLC conditions, gradient elution programs, and MS detection parameters were set according to our previous work (Wu et al. 2019) (link), described in Supplementary Material 1. To avoid bias and ensure the stability, samples were randomized in injection sequence, and the quality control (QC) samples which were prepared by pooling each serum sample before extraction, were inserted were tested at regular intervals throughout the sequence (Boretti et al. 2020) (link). The raw data were processed by Xcalibur 2.2 (Thermo-Fisher Scientific Inc.) and SIEVE 2.0 (Thermo-Fisher Scientific Inc.). The identification of lipid molecules was executed with the help of LIPIDMAPS (Sud et al. 2007 (link)) as well as the comparison of standards synthesized in our laboratory previously (Hui et al. 2003 (link)(Hui et al. , 2010)) (link). The amount of each lipid species was calculated by the IS amount and the peak area ratio of analyte/IS, as shown in Supplementary Material 2.
9
Alfaxalone Pharmacokinetics in Anesthetized Horses
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Blood samples were collected from 10 out of 25 horses in Group AM after induction of anesthesia; at 15, 30, and 45 min after connection to the breathing circuit; and immediately after standing. All blood samples were immediately placed on ice, and then the plasma was separated from the blood and frozen at − 20 °C. Alfaxalone in plasma was extracted by liquid–liquid extraction using methyl tert-butyl ether. The extracted substance was analyzed using liquid chromatography–tandem mass spectrometry consisted of Shimadzu prominence HPLC system (Shimadzu Co., Tokyo, Japan) and AB Sciex QTRAP 4000 mass spectrometer (AB Sciex, Framingham, MA, USA).
10
Quantification of Adenosine by HPLC-MS/MS
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A Prominence HPLC system (Shimadzu, Japan) equipped with a 3200 QTRAP MS/MS system with a Turbo V source and an ESI probe (AB SCIEX, Canada) was used. The HPLC separations were performed on a Tosoh TSKgel Amide-80 column (3 μm, 150 × 2.0 mm i.d.) and a Tosoh TSKgel ODS-100 V column (5 μm, 150 × 2.0 mm i.d.) with a mobile phase consisting of water and acetonitrile (linear gradient: 100 -90% acetonitrile over 12 min followed by 90 -30% over 8 min for the HILIC separation, and 0 -4% acetonitrile over 20 min for the ODS separation) at a flow rate of 0.2 mL min -1 at 40°C. Multiple-reaction monitoring (MRM) was used for the detection of adenosine (m/z: 268.1/136.1) and 15 N5-adenosine (m/z: 273.1/141.1) under positive ESI conditions.
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