Abstract
This chapter discusses the preparation and use of affinity columns with bovine milk folate-binding protein (FBP) covalently linked to sepharose 4B. Folates exist in biological tissues and fluids in microgram amounts, and their isolation in pure form has been difficult and time-consuming. The method discussed in this chapter utilizes affinity chromatography, with specific bovine milk FBP, with affinity for both mono- and polyglutamate folates. The binder is prepared from dried acid whey, by extraction, with K2HPO4, and subsequent purification is accomplished on a methotrexate-sepharose 4B column. This preparation is then fixed to activated sepharose 4B and used for the purification of biological folates on the basis of tight association at neutral pH that permits the removal of contaminant with neutralizer solution of high salt, and then elution of folate from binding protein at acid pH and elevated temperature. In this chapter, the purification of FBP from dried acid whey is discussed in detail. An overview of FBP assay is also discussed in the chapter. Fixation of FBP to activated sepharose 4B is described in this chapter. Purification of biological folate is discussed as well.
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