Separation of Bone NCP Proteins Binding to Hydroxyapatite

Methods: One gram of bone powder was prepared in freezer mill from 1 year bovine calf thigh bone. The bone powder was defatted for 24 hours in 1:1 acetone to ethanol and washed for 24 hours in 1M NaCl both stirred at 4°C. The powder was demineralized by stirring (3days) in 60ml of .5M EDTA, pH 7.5, 4°C, with protease inhibitors with decanting, collecting and refreshing daily 40 ml of solution. The collected protein solution was filtered through four filters, dialyzed and vortexed with .5 grams of hydroxyapatite powder (Bio-Gel HTP) per 13ml for 1 hour all at 4°C. The mixture was centrifuged 15 minutes at 4°C, RCF= 6000 and the tubes decanted and vortexed at 4°C with 1M NaCl for 15 minutes, centrifuged and decanted again. The tubes were vortexed 1 hour with .5M NaP04 buffer, pH 6.8, centrifuged as before and the decanted solution deionized and concentrated in Amicon ultra centrifuge filter tubes 10⁴ MWCO from approximately 200 ml to 180 µl. The concentrated protein was electrophoresed with SDS PAGE on 7.5% Tris-HCl Ready Gels and stained with bio-safe Coomassie. Results: Five protein bands stained the gel that had bound to hydroxyapatite. The molecular weights of these proteins were approximately 83kDa, 70kDa, 59kDa, 55kDa, and 45kDa. The strongest band was the 45kDa and the weakest was the 83kDa. The 83kDa and 55kDa bands may be bone sialoprotein. Conclusion: Vortexing of protein solutions with hydroxyapatite powder followed by vortex washing, debinding solutions and centrifugation can be substituted for column chromatography separation. Supported by AD Williams Grants