Isolation of phosphorated and non-phosphorylated osteopontin by immobilized metal (Fe+ ) affinity chromatography

To investigate osteopontin of differing degrees of phosphorylation, we used iron affinity chromatography to separate phosphorylated (pOPN) and non-phosphorylated OPN (npOPN). pOPN binds to ferric ions immobilized on metal chelating resins and can be eluted by increasing pH. The strength of binding depends riostly on serine phosphate content, thus making this a good method for isolating isoformsof OPN. Mouse epidermal JB6 Cl 41.5a cells were radiolabeled with 32PO4 or [ S]methionine after 24 hours treatment with TPA (yields pOPN) or calcitriol (yields npOPN). Total proteins were obtained by TCA precipitation and OPN was obtained by immunoadsorption assay. Samples were applied to columns packed with iron-loaded metal chelating resin and fractions eluted with buffers of increasing pH. Fractions eluting at pH 8.0 and 8.9 were pooled, desalted, concentrated and analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that pOPN was isolated with good selectivity.