0 50 ml polybuffer 74 pharmacia 350 ml dh 2 o u2212 ph to 4 0 with hcl 8 collect 2 ml fractions and test the ph of each fraction once the ph has dropped to 3 95 stop collecting fractions test the fractions using absorbance of 280 nm or by cytotoxicity on vero cells 10 fold dilutions 9 pool peak fractions and return ph to 7 0 using 1n naoh 10 clean column with 200 ml 1m nacl if dirty follow with 100 ml 1m hcl but quickly equilibrate column with 0 025m imidazole otherwise equilibrate with 24 etoh h 2 o 11 equilibrate cibachron blue 2 cm diameter 82 ml volume pierce with 100 ml of 10 mm sodium phosphate buffer wash buffer 12 load sample and follow with 60 ml of wash buffer 13 elute with 0 5m nacl in wash buffer and collect 2 ml fractions 14 test fractions for absorbance at 280 nm using the elution buffer as a blank and cytotoxicity on vero cells and pool appropriate fractions 15 clean column with 25 ml each of 8m urea in wash buffer and 1m nacl in wash buffer 16 reequilibrate column with 100 ml of wash buffer and add azide to the top of the column for storage 17 dialyse peak fractions against 4 l 0 01m tris cl ph 7 0 sigma 18 lyophilize sample and resuspend in 1 2 ml.