Purification of Peroxidase by HPLC on Concanavalin A Coupled to TSKgel® Tresyl-5PW

Purification of Peroxidase by HPLC on Concanavalin A Coupled to TSKgel® Tresyl-5PW

Conditions

column TSKgel Tresyl-5PW, 4 cm x 6 mm I.D., 10 µm particles (814455)
gradient step gradient at 4 min to 25 mM a-methyl-D-glucoside in binding buffer ((0.05 M acetate buffer, pH 5.0, containing 0.5 M NaCl and 1 mM each calcium chloride, manganese(II) chloride, and magnesium chloride))
flow rate 1 mL/min
sample crude peroxidase, 0.5 mg
detector Vis, 403 nm

Description

Analysis Note The following procedure is followed to prepare the Tresyl-5PW column and elute the active peroxidase fraction:

prepare ligand solution by dissolving 40 mg concanavalin A in 10 mL 0.1 M sodium bicarbonate, pH 8 containing 0.5 M NaCl;
washing step: wash column with deionized water;
coupling step: recycle the ligand solution overnight through the column at 0.2 mL/min and 25 °C (this creates the concanavalin A-modified column);
blocking step: block the residual tresyl groups with 0.1 M Tris-HCl, pH 8.0, at 1 mL/min for 1 hr at 25 °C;
binding buffer: 0.05 M acetate buffer, pH 5.0, containing 0.5 M NaCl and 1 mM each calcium chloride, manganese(II) chloride, and magnesium chloride;
elution step: step gradient at 4 min to 25 mM a-methyl-D-glucoside in binding buffer
Categories Analytical Chromatography, Proteins
Featured Industry Life Science and Biopharma
Legal Information TSKgel is a registered trademark of Tosoh Corporation
Other Notes Data provided by Tosoh Bioscience LLC.
suitability application for HPLC

Materials

     
Related Links