Extracellular non-collagenous bone proteins bind to hydroxyapatite(HAP) and affect bone mineralization. Objective: To compare the binding of different commercial HAP crystals for separating and purifying bovine bone proteins. Methods: Yearling bovine bone was spex milled to powder, defatted in 1:1 acetone:ethanol, and washed in 1M NaCl at 4°C for 24hrs. The powder was demineralized by stirring 72 hrs. in 0.5M EDTA(pH7.5) with protease inhibitors while decanting, collecting, and refreshing the solution daily. The protein solution was filtered, dialyzed, and divided into two equal volumes. Volume one was vortexed with Bio-Gel HTP powder for 1hr. at 4°C and volume two was vortexed with HAP Nanocrystals under equal conditions. Each volume was centrifuged 15min. at 4°C, RCF=6000, decant discarded, and vortexed with 10ml of 1M NaCl for 15min., then centrifuged and decant discarded again. The centrifugate was vortexed 1hr. with 0.5M NaPO4 (buffered pH6.8) centrifuged, decanted, and the solution was then deionized and concentrated in Amicon ultra-centrifuge filter tubes 104MWCO. The concentrated proteins were heated at 95°C for 5min. with ß-Mercapthonal, and electrophoresed with SDS PAGE . Group one proteins were electrophoresed on 7.5% Tris-HCl Ready-Gel and group two was electrophoresed on 4-15% Tris-HCl Ready-Gel. Pre-stained protein standard provided molecular weight reference. Kodak 1D-gel software captured protein bands and analyzed their molecular weights. Results: Nine protein bands ranged from 84.50kd(±1.76) to 26.75kd(±.52) in the Bio-Gel HTP group on 7.5% gel. Thirteen bands ranged from 87.19kd(±1.17) to 13.53kd(±.70) for the HAP Nanocrystals group on 4-15% gel . The nine protein bands from the Biogel HTP group correspond to the first nine bands for HAP Nanoparticles within their respective standard deviations. Conclusion: Bio-Gel HTP and HAP Nanoparticle offer controlled binding and releasing of bone proteins for separation and purification. The 4-15% Ready-Gel allowed separation of bone proteins at lower molecular weights.

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