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Purification and characterization of osteopontin from human milk.

Authors
  • Sørensen, Steen
  • Justesen, Steen Just
  • Johnsen, Anders H
Type
Published Article
Journal
Protein expression and purification
Publication Date
Aug 01, 2003
Volume
30
Issue
2
Pages
238–245
Identifiers
PMID: 12880773
Source
Medline
License
Unknown

Abstract

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.

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