Materials and Methods Column chroma

Materials and Methods Column chromatography with Ti-beads. We carried out cColumn chromatography was carried out as previously reported according to our previously reported methods (2 – 4),. We usedusungusing a column packed with Ti-beads (ø 45 µm, Osaka Ti Technologies, Co., Japan). Since According to company documantationdocumentation, the spherical Ti-beads were the productproduced by the gas atomizer method with and are spherical with have a , the surface of this right sphere material is the spheres is shining glittering smooth, shining y surface and all entirely covered with TiO2. We removed finer Finer particles were removed by particles by repeated decantation from a suspension in distilled water. We The beads were packed the beads into a commercial glass chromatography column equipped with a water jacket (XK16/20, GE Health Care, Tokyo, Japan) to obtain a bed volume of ø 16 × 50 mm. We washed theThe column was washed column with a large amount of dilute HCl (pH 4.0) and subsequently equilibrated with one of three different solvents:- dilute HCl at pH 4.0, 2 M urea in phosphate-buffered saline at pH 7.4 (PBS), or 2 M urea in dilute HCl at pH 4.0, respectively. We used These solvents were used for the following reasons: In standard acidic solutions such as dilute HCl (pH 3 – 4), type I collagen is primarily in a monomeric state but . Still, it often contains trace amounts of cross-linked polymers , which tend to aggregate under a slight change in the local environment, which may change their affinity with the Ti-column. We used 2 M urea was used because Mizuno and Hayashi (13) confirmed