Cells from 1 L cultures of RAB1 and RAB4R grown in LB for 30 h at 30 °C were pelleted at 6000× g for 15 min and supernatants were collected. The pH of the cell-free medium was adjusted to 3.0 by a slow addition of 6 N HCl and stirring on ice. LPs were allowed to precipitate overnight by incubation at 4 °C and pelleted by centrifugation at 10,000× g for 30 min. The pellets were dissolved initially in 20–40 mL of methanol and 10-fold concentrated by flash evaporation at room temperature. The crude LP preps were separated in a Dionex ICS-3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA), using a Protein and Peptide C18 column and a gradient 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 1 mL/min. Peptides eluting at all major peaks of absorbance were collected, concentrated by freeze-drying and dissolving in 0.5 mL of methanol and stored at −20 °C until further use.
Dionex ics 3000 hplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dionex ICS-3000 is a high-performance liquid chromatography (HPLC) system designed for the analysis of various ions and organic compounds. It features a modular design, allowing for flexible configuration to meet specific analytical requirements. The system includes a dual-pump module, a detector module, and a chromatography compartment with temperature control.
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Lab products found in correlation
2 protocols using dionex ics 3000 hplc system
1
Purification and Separation of Lipopolysaccharides
2
Size Exclusion Chromatography of Protein Samples
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BSA and BSA-Tpp samples containing 40 μg of protein were suspended in 25 mM phosphate buffer (pH 6.8) and centrifuged at 10 000 × g. The supernatant was transferred into HPLC vials and placed into cooled (4 °C) auto sampler chamber of HPLC system.
Size exclusion chromatography was performed using Dionex ICS-3000HPLC system (Thermo Scientific, USA) and the BioSep-SEC-S3000 (Phenomenex, 5 μm × 400 Å, 600 × 7.8 mm) column. The proteins were eluted at a flow rate 0.5 ml min−1 at 20 °C in isocratic 25 mM phosphate buffer (pH 6.8) containing 100 mM NaCl during 60 min. The column eluate was monitored by PDA detector at 280 and 420 nm with simultaneous spectra recording between 200 and 600 nm.
Size exclusion chromatography was performed using Dionex ICS-3000HPLC system (Thermo Scientific, USA) and the BioSep-SEC-S3000 (Phenomenex, 5 μm × 400 Å, 600 × 7.8 mm) column. The proteins were eluted at a flow rate 0.5 ml min−1 at 20 °C in isocratic 25 mM phosphate buffer (pH 6.8) containing 100 mM NaCl during 60 min. The column eluate was monitored by PDA detector at 280 and 420 nm with simultaneous spectra recording between 200 and 600 nm.
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