Prominence hplc system

Manufactured by Phenomenex

Sourced in United States

The Prominence HPLC system is a high-performance liquid chromatography system designed for reliable and efficient separation and analysis of a wide range of chemical compounds. It features a robust and precise solvent delivery system, a sensitive and versatile UV-Vis detector, and user-friendly software for method development and data analysis.

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Lab products found in correlation

Equinox 55 s ft ir spectrophotometer, by Bruker (5 mentions) Maxis 4g q tof mass spectrometer, by Bruker (4 mentions) Luna c18 column, by Phenomenex (4 mentions) 241 polarimeter, by PerkinElmer (3 mentions) Avance 600 mhz spectrometer, by Bruker (3 mentions) Acquity uplc system, by Bruker (3 mentions) Gemini c18 column, by Phenomenex (2 mentions) Kinetex 2.6 μmxb c18 100 column, by Phenomenex (1 mentions) Biosep sec s3000 column, by Phenomenex (1 mentions) Labsolutions software, by Shimadzu (1 mentions) Avance 500 mhz spectrometer, by Bruker (1 mentions) Luna phenyl hexyl analytical column, by Phenomenex (1 mentions) Ultimate 3000 semiprep hplc, by Thermo Fisher Scientific (1 mentions) Jupiter proteo c 12 column, by Phenomenex (1 mentions) Kinetex 5 m c18 column, by Phenomenex (1 mentions) Avance 3 500 mhz, by Bruker (1 mentions) Avance 3 hd 400 mhz, by Bruker (1 mentions) Maxis 4g esi qtof, by Bruker (1 mentions) Luna c18 2, by Phenomenex (1 mentions) Microtof 2 mass spectrometer, by Bruker (1 mentions)

15 protocols using prominence hplc system

Quantification of HDAC11 Inhibitors

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The determination of kinetic constants
and IC₅₀ values of inhibitors was carried out in parallel
to the continuous fluorescence assay by discontinuous assays analyzed
by means of reversed-phase high-performance liquid chromatography
(RP-HPLC). The reaction buffer, concentration of enzyme, substrates,
and inhibitors were carried out as described above. The reaction was
quenched by the addition of 0.5% acetic acid after 30 min of incubation
and centrifuged at 2000g at 37 °C for 15 min
to remove precipitated BSA and HDAC11. The reactions were analyzed
by RP-HPLC (Shimadzu, HPLC Prominence system) with a Kinetex 2.6 μm
XB-C18 100 Å column (100 × 3 mm; Phenomenex, Torrance, CA).
The mobile phase A was 5% acetonitrile with 0.1% (v/v) TFA and the
mobile phase B was 95% acetonitrile with 0.1% (v/v) TFA. The separation
of the reaction product from the acylated substrate was performed
in a 12-min linear gradient from 10 to 60% of eluent B at a flow rate
of 0.6 mL/min. The product and substrate peaks were quantified using
the absorbance at 365 nm (absorption of the 3-nitrotyrosyl moiety)
to verify the results of the fluorescence assay.

Kutil Z., Mikešová J., Zessin M., Meleshin M., Nováková Z., Alquicer G., Kozikowski A., Sippl W., Bařinka C, & Schutkowski M. (2019). Continuous Activity Assay for HDAC11 Enabling Reevaluation of HDAC Inhibitors. ACS Omega, 4(22), 19895-19904.

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Fluorophore-labeled Peptide HDAC6 Assay

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Individual fluorophore-labeled peptides were incubated with HDAC6 at 37 °C for 30 min in an assay buffer comprised of 50 mM HEPES, 140 mM NaCl, 10 mM KCl, 2 mg/ml BSA, and 1 mM TCEP, at a pH 7.4 adjusted with NaOH. The reaction was quenched by the addition of acetic acid to a final concentration of 0.5%, and reaction products were quantified by means of RP-HPLC (Shimadzu, HPLC Prominence system) on the column Kinetex® 2.6 µm XB-C18 100 Å, 100 × 3 mm (Phenomenex, Torrance, CA, USA).

Skultetyova L., Ustinova K., Kutil Z., Novakova Z., Pavlicek J., Mikesova J., Trapl D., Baranova P., Havlinova B., Hubalek M., Lansky Z, & Barinka C. (2017). Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules. Scientific Reports, 7, 11547.

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Fluorescent Peptide HDAC11 Deacetylation Assay

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Individual Abz-labeled fluorescent peptides (10 µM) were incubated with 100 nM HDAC11 at 37°C for 30 mins. in an assay buffer comprising 50 mM HEPES, 140 mM NaCl, 10 mM KCl, 2 mg/ml BSA, 1 mM TCEP, pH 7.4. The reaction was terminated by the addition of acetic acid to a final concentration of 0.5 % and the reaction products quantified by means of RP-HPLC (Shimadzu, HPLC Prominence system) on a column Kinetex® 2.6 µm XB-C18 100 Å, 100 x 3 mm (Phenomenex, Torrance, CA, USA). The fluorescence detection with excitation/emission wavelengths set at 320/420 nm, respectively, was used together with a calibration curve of known concentrations of the fluorescent reaction product.

Kutil Z., Novakova Z., Meleshin M., Mikesova J., Schutkowski M, & Barinka C. (2018). Histone Deacetylase 11 Is a Fatty-Acid Deacylase. ACS chemical biology, 13(3).

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RL71 Extraction and Quantification from Tissues

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In order to extract RL71 from the various tissues, 50 mg of ground tissue was mixed with 0.5 mL of methanol, sonicated and incubated for 1 h on ice. The homogenate was then centrifuged at 14,000 rpm for 10 min and the supernatant reduced down to 100 µL using low-flow nitrogen to evaporate the methanol. Samples were centrifuged again and transferred to a high-performance liquid chromatography (HPLC) vial. The analysis was performed on a Shimadzu Prominence HPLC system using Chromeleon software and a Phenomenex Gemini^® 5 µm C18 110 Å LC Column 150×4.6 mm. A flow rate of 1 mL/min was used with an 80 µL injection volume. Peaks were eluted with 10 mM sodium acetate pH 5.0 as mobile phase A and acetonitrile as mobile phase B. An isocratic flow of 35% A and 65% B was maintained over 10 min. The eluted peak for RL71 was monitored at 361 nm with a retention time of 4.4 min.

Martey O., Nimick M., Taurin S., Sundararajan V., Greish K, & Rosengren R.J. (2017). Styrene maleic acid-encapsulated RL71 micelles suppress tumor growth in a murine xenograft model of triple negative breast cancer. International Journal of Nanomedicine, 12, 7225-7237.

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Spectroscopic Analysis of Organic Compounds

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Optical rotations were measured on a Perkin–Elmer 241 Polarimeter. UV spectra were recorded on an Aminco/OLIS UV-Vis Spectrophotometer. ECD spectra were recorded on an AVIV Model 420 Circular Dichroism Spectrometer. IR spectra were measured with a Bruker Equinox 55/S FT–IR Spectrophotometer. Both 1D and 2D NMR spectra were obtained using a Bruker Avance 600 MHz spectrometer with ¹H{¹³C/¹⁵N} cryoprobe and a 500 MHz spectrometer with ¹³C/¹⁵N{¹H} cryoprobe; chemical shifts were referenced to the residual solvent peaks (CD₃OD: δ_H = 3.31, δ_C = 49.15). HRMS and MS/MS data were acquired with a Bruker MaXis 4G QTOF mass spectrometer. RP HPLC was performed using a Shimadzu Prominence HPLC system and a Phenomenex Luna C₁₈ column (250 × 10 mm, 5 µm).

Zhang F., Braun D.R., Chanana S., Rajski S.R, & Bugni T.S. (2019). Phallusialides A−E, Pyrrole-Derived Alkaloids Discovered from a Marine-derived Micromonospora sp. Bacterium Using MS-based Metabolomics Approaches. Journal of natural products, 82(12), 3432-3439.

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Peptide-MHC I Complex Exchange Assay

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To initiate peptide exchange, 0.5 µM peptide–MHC I complex was incubated with 50 µM exchange peptide in 110 µl PBS under defined exchange conditions. After incubation exchange solutions were centrifuged at 14,000 g for 1 min at room temperature, and subsequently, the supernatant was analyzed by gel filtration on a Shimadzu Prominence HPLC system equipped with a 300 × 7.8 mm BioSep SEC–s3000 column (Phenomenex) using PBS as mobile phase. Data were processed and analyzed using Shimadzu LabSolutions software (version 5.85).

Luimstra J.J., Garstka M.A., Roex M.C., Redeker A., Janssen G.M., van Veelen P.A., Arens R., Falkenburg J.H., Neefjes J, & Ovaa H. (2018). A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells. The Journal of Experimental Medicine, 215(5), 1493-1504.

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Spectroscopic Characterization of Compounds

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UV spectra were recorded on an Aminco/OLIS UV-Vis Spectrophotometer (Bogart, GA, USA). IR spectra were measured with a Bruker Equinox 55/S FT–IR Spectrophotometer (Santa Barbara, CA, USA). Both 1D and 2D NMR spectra were obtained using a Bruker Avance 500 MHz spectrometer (Billerica, MA, USA) with ¹H{¹³C/¹⁵N} cryoprobe and a 500 MHz spectrometer with ¹³C/¹⁵N{¹H} cryoprobe; chemical shifts were referenced to the residual solvent peaks (CD₃OD: δ_H = 3.31, δ_C = 49.15; DMSO-d₆: δ_H = 2.50, δ_C = 39.51). HRMS data were acquired with a Bruker MaXis 4G QTOF mass spectrometer (Billerica, MA, USA). RP HPLC was performed using a Shimadzu Prominence HPLC system and a Phenomenex Luna C₁₈ column (250 × 10 mm, 5 µm) (Torrance, CA, USA).

Zhang F., Braun D.R., Rajski S.R., DeMaria D, & Bugni T.S. (2019). Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium Enhygromyxa sp.. Marine Drugs, 17(12), 698.

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Comprehensive Analytical Characterization Protocol

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Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UV spectra were recorded with an Aminco/OLIS UV-Vis spectrophotometer. IR spectra were measured with a Bruker Equinox 55/S FT-IR spectrophotometer. 1D and 2D NMR data were recorded using a Bruker Avance 600 MHz spectrometer with a ¹H{¹³C/¹⁵N} cryoprobe and a 500 MHz spectrometer with a ¹³C/¹⁵N{¹H} cryoprobe, AVANCE-500, or DRX-400 spectrometers. Chemical shift values were referenced to the residual solvent peaks (CDCl₃: δ_H7.26, δ_C 77.18; methanol-d₄: δ_H 3.31, δ_C 49.15) HRMS data were acquired with a Bruker Maxis 4G QTOF mass spectrometer. Reverse phase (RP) HPLC was performed using a Shimadzu Prominence HPLC System and a Phenomenex Luna C-18 semi-prep column (250 × 10 mm, 5 μm), Phenomenex Luna phenyl-hexyl semi-prep column (250 × 10 mm, 5 μm) or a Phenomenex Luna phenyl-hexyl analytical column (250 × 4.6 mm, 5 μm).

Jayanetti D.R., Braun D.R., Barns K.J., Rajski S.R, & Bugni T.S. (2019). Bulbiferates A and B: Antibacterial Acetamidohydroxybenzoates from a Marine Proteobacterium, Microbulbifer sp.. Journal of natural products, 82(7), 1930-1934.

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Purification of Peptides and Conjugates

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The purification of crude peptides and conjugates was performed on an UltiMate 3000 Semiprep HPLC (Thermo Fisher Scientific, Waltham, MA, USA) with a Phenomenex Jupiter Proteo C-12 column (250 × 10 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).
The crude and purified peptides were analyzed by analytical RP-HPLC (Shimadzu prominence HPLC system) with a Phenomenex Jupiter Proteo C-12 column (150 × 4.6 mm) using gradient elution, consisting of 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile/water = 80/20 (v/v) (eluent B).

Szabó I., Biri-Kovács B., Vári B., Ranđelović I., Vári-Mező D., Juhász É., Halmos G., Bősze S., Tóvári J, & Mező G. (2024). Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH–Peptide Conjugates. International Journal of Molecular Sciences, 25(2), 1095.

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RP-HPLC Analysis of Compounds FL-1 and FL-2

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Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses were performed on a Shimadzu Prominence HPLC system (Kyoto, Japan) using a Kinetex 5 µm C-18 column (100 Å, 250 × 4.6 mm column, Phenomenex). All analyses were performed at ambient temperature. The HPLC conditions were as follows: buffer A: 0.1 M CH₃COONH₄, pH 6.7/H₂O; buffer B: CH₃CN; and flow rate: 1 mL min-1. The gradient of buffer B was as follows: 0→2 min 0%; 2→25 min 0–45%; 25→28 min 45–60%; 28→30 min 60–0%; 30→33 min 0% for FL-1 (retention time, RT = 14.3 min) and FL-2 (RT = 13.7 min). UV detection was performed at λmax 260 nm and λmax 494 nm, and the amount of compound was defined in optical units (OD) at the 260 nm wavelength.

Kaniowski D., Ebenryter-Olbińska K., Kulik K., Suwara J., Cypryk W., Jakóbik-Kolon A., Leśnikowski Z, & Nawrot B. (2021). Composites of Nucleic Acids and Boron Clusters (C2B10H12) as Functional Nanoparticles for Downregulation of EGFR Oncogene in Cancer Cells. International Journal of Molecular Sciences, 22(9), 4863.

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