Heparin sepharose 6 fast flow

25 mL human serum from freshly drawn blood was pumped (0.5 mL/min) through a column filled with 5 mL heparin immobilized adsorbent (Heparin Sepharose 6 fast flow, GE Healthcare, Chicago, USA). To remove all non-heparin-binding substances from the adsorbent surface, the column was rinsed with 10 mL physiological saline solution. The heparin-bound plasma components were eluted from the column in two steps. Using 10 mL of 0.5 M NaCl solution, first those substances with a low to medium binding constant to heparin were eluted from the column. Substances strongly bound to heparin were finally eluted with 10 mL 2.2 M NaCl solution. The eluates were separated into 1 mL fractions using a fraction collector from (Bio-Rad, California, USA). Protein concentration of each fractions was analyzed with a Cobas c311 analyzer from Roche (Basel, Switzerland) with according test reagents. Fractions of each salt concentration were pooled and desalinated and concentrated with VIVASPIN 20 with a molecular cut off of 3 kDa (Sartorius, Göttingen, Germany). The concentrate of each fraction was washed once and finally diluted to a volume of 1.5 mL with ringer solution. Finally, 1:10 with ringer solution diluted serum and the two heparin-binding serum fractions (0.5, 2.2 M NaCl) were spiked with 5 ng/mL LPS (E. coli) and their endotoxin activity was determined by the LAL test.

BMP-2 refolding was carried out by dilution of protein in refolding buffer (100 mM Tris/HCl pH=8.3, 5 mM EDTA, 250 mM NaCl, 0.5 M L-arginine) to decrease protein concentration and prevent aggregation. Two oxidoreductase systems (reduced glutathione/ oxidized glutathione and Cysteine/Cystine) and different refolding methods were used to analyze disulfide bond formation as well as protein dimerization (Table 1). BMP-2 refolding was started by gradual addition of denatured protein to pre-cooled refolding buffer until the concentration fell below 0.2 mg/ml while stirring at 4°C for 24 hr. The protein concentration increased to 1 mg/ml by cross flow filtration (Amicon ultra centrifugal filter) and redox system components were removed by dialysis against urea 6 M, 0.1 M Tris/HCl and 5 mm EDTA, pH=6. Dimeric protein was eluted by heparin column (Heparin sepharose ^™ 6 Fast Flow, GEHealthcare, 17-0998-01) at 0.3 and 0.7 M NaCl and purified protein was dialyzed against 20 mM ammonium acetate buffer, pH=4.8 and lyophilized.