4.3. Affinity Chromatography on a Heparin Column

Protein purification of whole plant extracts was conducted using the ÄKTA Explorer System (Amersham Biosciences). Seven samples showing nucleolytic activity were run over a column filled with heparin (HiTrap Heparin column, 0.7·2.5 cm; GE Healthcare, cat. no. 17-0406-01). Heparin shows high affinity to DNA-binding proteins; hence, it is the best choice for affinity chromatography of nucleic acid binding proteins. Proteins with high affinity for heparin were bound to the resin, while the other components were carried through in buffer A and collected in “flow-through” fraction nos. 1–10. The proteins of interest were returned to solution in buffer B in fractions ~18–40. Absorbance was measured at 280 nm for all fractions, and chromatographs were plotted by UNICORN V3.0 software, Amersham Biosciences UK Limited, Little Chalfont, UK. For each run, 40 to 44 fractions were collected.

To isolate and purify proteins from the milky sap, approximately 0.5 μg protein was loaded onto a HiTrap heparin column equilibrated with 0.1 M Tris-HCl, pH 8.0, containing 10% glycerol. The column was eluted with a linear gradient of 0 to 2 M NaCl in the same buffer. The absorbance at 280 nm and DNase activity of all fractions (volume 1 mL) were determined.