Abstract
The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.
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Acknowledgment
This work was supported by the National Institute of Dental and Craniofacial Research (NIDCR, US National Institutes of Health [NIH]) grant DE018020, and JSPS KAKENHI, Grant-in-Aid for Scientific Research (C; 26462982).
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Yamakoshi, Y., Hu, J.CC., Saito, M.M., Simmer, J.P. (2019). Protocols for Studying Formation and Mineralization of Dental Tissues In Vivo: Extraction Protocol for Isolating Dentin Matrix Proteins from Developing Teeth. In: Papagerakis, P. (eds) Odontogenesis. Methods in Molecular Biology, vol 1922. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9012-2_24
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DOI: https://doi.org/10.1007/978-1-4939-9012-2_24
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