Purification of DNA Binding Protein using Heparin Sepharose

Performing a separation Heparin Sepharose 6 Fast Flow, Heparin Sepharose High Performance

Binding buffer: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0 Elution buffer: 20 mM Tris-HCl, 1 to 2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1 to 2 M NaCl, pH 7.0 Equilibrate the column with 10 CV of binding buffer. Apply the sample. Wash with 5 to 10 CV of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm). Elute with 5 to 10 CV of elution buffer using a continuous or step gradient from 0% to 100% elution buffer. Modify the selectivity of heparin by altering pH or ionic strength of the buffers. Elute using a continuous or step gradient with NaCl, KCl or (NH4)2SO4 up to 2 M.

If used for purification or removal of coagulation factors:

Since the heparin acts as an affinity ligand for coagulation factors, it is advisable to include a minimum concentration of 150 mM NaCl in the binding buffer.

If an increasing salt gradient gives unsatisfactory results, use heparin (1 to 5 mg/ml) as a competing agent in the elution buffer.

Cleaning Remove ionically bound proteins by washing with 0.5 CV of 2 M NaCl for 10 to 15 min.

Remove precipitated or denatured proteins by washing with 4 CV of 100 mM NaOH for 1 to 2 h; or 2 CV of 6 M guanidine hydrochloride for 30 to 60 min; or 2 CV of 8 M urea for 30 to 60 min. Remove hydrophobically bound proteins by washing with 4 CV of 0.1% to 0.5% Tween 20 for 1 to 2 h.

Chemical stability 100 mM NaOH (1 w at 20°C), 50 mM sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride.