Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 30, Issue 2, Pages 238-245Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S1046-5928(03)00102-5
Keywords
chromatography; crystallization; amino acid sequence; phosphorylation
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Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M-r OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies. (C) 2003 Elsevier Science (USA). All rights reserved.
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