Analytical size-exclusion HPLC was performed for purified chimeric IL-15 by using the Agilent 1260 Infinity instrument equipped with a UV detector to further confirm the presence of dimeric forms. About 20 µl of 1 μg/ml chimeric IL-15 diluted into PBS was injected into a Zorbax Bio Series GF-250 column with dimensions of 9.4 (i.d.) × 250 mm (Agilent Technologies). Phosphate-buffered saline was used as the mobile phase and a 1 ml/min flow rate was maintained. The programme was run for 45 min. OVA and BSA were used as standards. The chromatograms were analyzed by the open lab (Agilent) and presented in form of overlap peaks of chimeric IL-15, OVA and BSA.
Zorbax bio series gf 250 column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax Bio Series GF-250 column is a size exclusion chromatography column designed for the separation and purification of biomolecules. It is capable of separating proteins, peptides, and other macromolecules based on their size and molecular weight.
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4 protocols using zorbax bio series gf 250 column
1
Size-exclusion HPLC Characterization of Chimeric IL-15
2
FLAG-Tagged Protein Purification by FPLC
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HEK 293T cells transfected with ODC-FLAG or AZIN2-FLAG were lysed in solubilization buffer and centrifuged at 14.000×g for 20 min, being the supernatant directly injected into a Zorbax Bio Series GF-250 column (Agilent Technologies, CA, USA) in a buffer containing 50 mM Tris, 1 mM EDTA, 4 mM pyridoxal phosphate, and 0.1% Igepal. Sixty 100-μl fractions were collected and analyzed by Western blotting and incubation with anti-FLAG antibody. Bovine serum albumin (Mr 66,000) was used as standard, but in this case fractions were separated by SDS–PAGE and stained with Commasie Blue.
3
HPLC Analysis of Lysozyme Proteins
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HPLC (High-Performance Liquid Chromatography) studies were performed with Agilent 1100 Series chain equipped with an UV detector and a quaternary pump. The column used for separation of proteins in the range of 400 000 to 4000 g mol−1 was a 9.4 × 250 mm Zorbax Bio Series GF-250 column (Agilent).
The buffer solutions for the mobile phase were prepared by dissolving 1 tablet of phosphate buffer saline (PBS from Sigma-Aldrich), 0.1% wt sodium dodecyl sulfate (SDS purchased from SIGMA-ALDRICH) and 0.005% wt sodium azide (NaN3) in 200 mL of demineralized water. The pH was 7.4 at 25 °C. The concentrations were 0.01 mol L−1 for phosphate buffer, 0.0027 mol L−1 for potassium chloride and 0.137 mol L−1 for sodium chloride.
100 µL of lysozyme solution was injected in the column and analyzed at the wavelength of 280 nm. The analysis was performed for 15 min, with a flow rate of 1.0 mL min−1 and a constant temperature of 25 °C.
The buffer solutions for the mobile phase were prepared by dissolving 1 tablet of phosphate buffer saline (PBS from Sigma-Aldrich), 0.1% wt sodium dodecyl sulfate (SDS purchased from SIGMA-ALDRICH) and 0.005% wt sodium azide (NaN3) in 200 mL of demineralized water. The pH was 7.4 at 25 °C. The concentrations were 0.01 mol L−1 for phosphate buffer, 0.0027 mol L−1 for potassium chloride and 0.137 mol L−1 for sodium chloride.
100 µL of lysozyme solution was injected in the column and analyzed at the wavelength of 280 nm. The analysis was performed for 15 min, with a flow rate of 1.0 mL min−1 and a constant temperature of 25 °C.
4
HPLC Analysis of Lysozyme Proteins
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HPLC (High-Performance Liquid Chromatography) studies were performed with Agilent 1100 Series chain equipped with an UV detector and a quaternary pump. The column used for separation of proteins in the range of 400 000 to 4000 g.mol - 1 was a 9.4 x 250 mm Zorbax Bio Series GF-250 column (Agilent).
The buffer solutions for the mobile phase were prepared by dissolving 1 tablet of phosphate buffer saline (PBS from Sigma-Aldrich), 0.1% wt sodium dodecyl sulfate (SDS purchased from SIGMA-ALDRICH) and 0.005% wt sodium azide (NaN 3 ) in 200 mL of demineralized water. The pH is 7.4 at 25°C. The concentrations are 0.01 mol.L - 1 for phosphate buffer, 0.0027 mol.L - 1 for potassium chloride and 0.137 mol.L - 1 for sodium chloride. 100 µL of lysozyme solution was injected in the column and analyzed at the wavelength of 280 nm. The analysis was performed for 15 minutes, with a ow rate of 1.0 mL.min - 1 and a constant temperature of 25°C.
The buffer solutions for the mobile phase were prepared by dissolving 1 tablet of phosphate buffer saline (PBS from Sigma-Aldrich), 0.1% wt sodium dodecyl sulfate (SDS purchased from SIGMA-ALDRICH) and 0.005% wt sodium azide (NaN 3 ) in 200 mL of demineralized water. The pH is 7.4 at 25°C. The concentrations are 0.01 mol.L - 1 for phosphate buffer, 0.0027 mol.L - 1 for potassium chloride and 0.137 mol.L - 1 for sodium chloride. 100 µL of lysozyme solution was injected in the column and analyzed at the wavelength of 280 nm. The analysis was performed for 15 minutes, with a ow rate of 1.0 mL.min - 1 and a constant temperature of 25°C.
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