The extracellular matrix of the bone and dentin contains several non-collagenous proteins (NCPs). One category of NCPs is termed the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), etc. These proteins have abundant phosphoserines, aspartic acids, and glutamic acids. In this protocol, we describe the extraction of NCPs from the bone and dentin matrices using guanidine-HCl/EDTA and the isolation of polyanionic SIBLINGs from NCPs using ion-exchange fast protein liquid chromatography (FPLC) to separate the differentially charged proteins into different fractions through a gradient elution by NaCl.
Keywords: FPLC; Ion-exchange chromatography; N-linked glycoprotein; Non-collagenous protein; Small integrin-binding ligand; Stains-all staining; Ultrafiltration.