The culture broth was centrifuged at 7000 × g for 10 min, and the supernatant was filtered through a 0.22-μm pore filter (Nucleopore, Costar). A total of 100 μl of crude peptides was subjected to a reversed phase (RP) high-performance liquid chromatography system (HPLC; Agilent, USA) with a semi-preparative Zorbax 300SB-C8 column (250 mm × 9.4 mm, 5-μm particle size, 300-Å pore size) (Agilent, USA). The column was equilibrated in 0.1% (v/v) trifluoroacetic acid and 10% acetonitrile and then developed with a linear 0% to 60% acetonitrile gradient at a flow rate of 1.0 ml/min. The absorbances at 214 and 280 nm were monitored, and the peaks were investigated using an antimicrobial activity assay with E. coli serving as the indicator strain5 (link). The purified peptides were verified using an RP-HPLC analytical Zorbax 300SB-C8 column (250 mm × 4.6 mm, 5 μm, 300 Å) (Agilent, USA) and were freeze-dried in a vacuum freeze dryer (SIM International Group Co., Ltd., USA) at −80 °C for further experiments. The concentration of the purified peptide solutions was determined by UV spectrophotometry30 (link)31 (link). The molecular weight of the purified CAM-W was measured using electrospray ionization mass spectrometry (Agilent, USA).
Zorbax 300sb c8 column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax 300SB-C8 column is a reversed-phase liquid chromatography column designed for the separation and analysis of a wide range of biomolecules. It features a spherical silica-based stationary phase with a carbon-8 (C8) ligand. The column is suitable for use in applications such as the analysis of peptides, proteins, and other biomolecules.
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Lab products found in correlation
1200 sl lc system, by Agilent Technologies (3 mentions)
6520 q tof mass spectrometer, by Agilent Technologies (2 mentions)
Capillary lc nano esi ms, by Agilent Technologies (2 mentions)
Ziptip, by Merck Group (2 mentions)
6545 q tof lc ms, by Agilent Technologies (2 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Daltonics ultraflextreme, by Bruker (1 mentions)
Ltq ft ultra, by Thermo Fisher Scientific (1 mentions)
Ettan mdlc, by GE Healthcare (1 mentions)
Deconvolute ms protein, by Agilent Technologies (1 mentions)
Nucleopore, by Corning (1 mentions)
G1969 85000, by Agilent Technologies (1 mentions)
Agilent 6520 q tof mass spectrometer, by Agilent Technologies (1 mentions)
Rp hplc, by Agilent Technologies (1 mentions)
Sil 30ac, by Shimadzu (1 mentions)
Lc 20ap, by Shimadzu (1 mentions)
Zorbax 300sb c18 column, by Agilent Technologies (1 mentions)
Lc 30ad, by Shimadzu (1 mentions)
Luna c8 column, by Phenomenex (1 mentions)
31 protocols using zorbax 300sb c8 column
1
Purification and Characterization of Antimicrobial Peptide
2
Purification and Quantification of Sublancin 168
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Isolation and purification of sublancin 168 were carried out as previously described [1 (link)], with slight modification. The collected supernatant was made in 1 M NaCl and subjected to a hydrophobic interaction chromatography of 25 mL Toyopearl Butyl-650 column (Tosoh, Tokyo, Japan), and then a solution of 50 mM NaAc, pH 4.0, was used to wash down the sublancin. Subsequently the elution was made in 0.1 trifluoroacetic acid (TFA) and subjected to a semipreparative Zorbax 300SB-C8 column (250 × 9.4 mm, 5 μm particle size, 300 Å pore size) (Agilent, Englewood, CO) with a linear 0–60% acetonitrile gradient at a flow rate of 1.0 mL/min. The active fractions were collected and applied to an analytical Zorbax 300SB-C8 column (150 × 4.6 mm, 5 μm particle size, 300 Å pore size) (Agilent, Englewood, CO) with the same conditions as the first step. The absorbances at 214 nm, 254 nm, and 280 nm were monitored. The concentration of purified sublancin 168 was determined by UV spectrophotometry [13 (link), 14 (link)]. Using purified sublancin as standard sample, the fermentation broths were applied to analytical Zorbax 300SB-C8 column to determine sublancin 168 concentrations with the method used in purification of sublancin 168.
3
Peptide Synthesis and Characterization
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Peptides synthesis were carried out by solid phase peptide synthesis using Fmoc (9-fluorenyl-methoxycar-bonyl) chemistry and Rink amide 4-methylbenzhydrylamine resin (MBHA resin; 0.8 mmol/g), as described previously (Chen et al., 2005 (link); Huang et al., 2010a ). The crude peptides were purified by preparative Shimadzu LC-6A high-performance liquid chromatography (HPLC), using a Zorbax 300 SB-C8 column (250 × 9.4-mm ID, 6.5-mm particle size, 300-Å pore size; Agilent Technologies) with a linear AB gradient (0.1% acetonitrile/min) at a flow rate of 2 mL/min, while eluent A was 0.1% aqueous trifluoroacetic acid (TFA) in water, and eluent B was 0.1% TFA in acetonitrile. Peptide samples were analyzed on a Shimadzu LC-20A HPLC. Runs were performed on a Zorbax 300 SB-C8 column (150 × 4.6-mm ID, 5-mm particle size, 300-Å pore size) from Agilent Technologies, using a linear AB gradient (1% acetonitrile/min) and a flow rate of 1 mL/min, in which eluent A was 0.1% aqueous TFA and eluent B was 0.1% TFA in acetonitrile. The peptides were further characterized by mass spectrometry and amino acid analysis (Lee et al., 2003 (link); Mant et al., 2003 (link)).
4
Quantitative Analysis of Quercetin Metabolites
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EAT was homogenized with 0.1% phosphate buffer (pH 5.3). Quercetin and the metabolites were extracted with acetonitrile or ethyl acetate after treatment with sulfatase possessed β‐glucuronidase activity (Sigma–Aldrich, MO, USA), respectively, and then quantified as quercetin and isorhamnetin by the ACQUITY UPLC system connected to a XEVO TQD equipped with a Zspray ion source (Waters, MA, USA). Data acquisition and mass spectrometric evaluation were conducted using MassLynx software (ver. 4.1, Waters). The HPLC conditions were as follows: the ratio of acetonitrile in 0.1% formic acid was linearly increased from 10 to 50% over 15 min at 0.2 mL/min on a Zorbax300 SB‐C8 column (2.1 × 150 mm, 5 μm; Agilent Technologies) at 40°C. Capillary voltage (4 kV), source temperature (150°C), desolvation temperature (500°C), cone gas flow (50 L/h), and desolvation gas flow (800 L/h) were optimized for selected reaction monitoring (SRM) intensity. Cone voltage, collision energy, and the SRM transitions were 64 V, 30 V, and m/z 303/153 for quercetin; 56 V, 34 V, and m/z 317/153 for isorhamnetin; 32 V, 14 V, and m/z 479/303 for quercetin 3‐o’‐glucronide; 42 V, 22 V, and m/z 383/303 for quercetin 3‐o‐sulfate; and 66 V, 32 V, and m/z 275/154 for the internal standard, genistein‐d4, respectively.
5
Purity Analysis of Target Proteins
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The purity of the target proteins was analyzed by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC); the absorbance values at 280 nm were recorded. SEC-HPLC (TSK gel G2000SWXL, 5 μm, Ф7.8 × 300 mm) was performed using a mobile phase of 20 mmol·L−1 PB pH 7.0 containing 150 mmol·L−1 NaCl at a flow rate of 0.5 mL·min−1. RP-HPLC was performed using an Agilent ZORBAX 300SB-C8 column, 5 μm, Ф4.6 × 250 mm for analysis. The mobile phase A was 1‰ aqueous trifluoroacetic acid (TFA), and mobile phase B was1‰TFA in 95% acetonitrile at a flow rate of 1 mL·min−1, and the elution conditions were 0%–80% B over 40 min.
6
Mass Spectrometry Techniques for Protein Analysis
7
Protein Characterization by SDS-PAGE and HPLC-MS
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check protein purity and composition as previously described [32 (link)] using QuickRun buffer (MDbio) and protein standards ranging from 10 to 245 kDa or from 14 to 116 kDa (New England BioLabs). Protein quantification was performed using the Bradford method before further analyses [33 (link)]. Protein molecular weight was determined using a high-performance liquid chromatography (HPLC) system (Agilent 1290) coupled to a Q-TOF–MS (Agilent 6530). The HPLC was equipped with a Zorbax 300SB-C8 column (4.6 × 250 mm, Agilent). Samples were run at a flow rate of 1 mL/min with 6-μL injection volume. The mobile phases contained 0.1% (vol/vol) formic acid in water (A) or in acetonitrile (B). A four-step linear gradient of 5% B from 0 to 5 min, 5–50% B from 5 to 10 min, 50–90% B from 10 to 12 min, and 90% B from 12 to 15 min was used. The scan range was m/z 800–1800. The deconvolution analysis was displayed by the software Deconvolute (MS):protein (Agilent).
8
HPLC Purification of Compound P6
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High-performance liquid chromatography (HPLC) was applied to detect the purity of P6 using an Agilent series 1100 HPLC system connected to a ZORBAX®300SB-C8 column (4.6 × 150 mm, 5 µm; Agilent, Foster City, CA, USA). Water-trifluoroacetic acid (solvent A; 100:0.1, v/v) and acetonitrile-trifluoroacetic acid (solvent B; 100:0.1, v/v) were used as elution solvents. The elution procedure was set as follows: 55% solvent A and 45% solvent B with the flow rate at 1 mL/min. The wavelength of the UV detector was at 280 nm, and column temperature was 30 °C.
9
Quantitative LC-QTOF-MS Metabolomic Analysis
10
Lipid Extraction and LC-QTOF-MS Analysis
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For lipid extraction Elf1 and H1 cells were grown on matrigel for one passage. Cells were washed with PBS and 37 °C deionized water followed by the addition of 0.5ml of a −75 °C solution of internal standards54 (link) and incubation on dry ice for 15 min. Cells were scraped into eppendorf tubes and 1ml of chloroform was added, followed by 15 min incubation on dry ice and spun for 5 min at 4 °C at 18000 rcf, after which the lower phase was collected and stored at −80 °C.
LC-QTOF-MS experiments were performed using an Agilent 1200 SL LC system coupled online with an Agilent 6520 Q-TOF mass spectrometer. Each sample (4 µL for positive ESI ionization, 8 µL for negative ESI ionization) was injected onto an Agilent Zorbax 300 SB-C8 column, which was heated to 50 °C.
LC-QTOF-MS experiments were performed using an Agilent 1200 SL LC system coupled online with an Agilent 6520 Q-TOF mass spectrometer. Each sample (4 µL for positive ESI ionization, 8 µL for negative ESI ionization) was injected onto an Agilent Zorbax 300 SB-C8 column, which was heated to 50 °C.
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