Isolation of Native and Proteolytically Derived Ileal Receptor for Intrinsic Factor—Cobalamin

Bellur Seetharam, David H. Alpers

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

This chapter describes the assay method, purification, and isolation of native and proteolytically derived ileal receptor for intrinsic factor–cobalamin. Receptor activity during the purification was assessed by measuring the binding of the hog IF-[57Co]Cbl complex made with purified hog intrinsic factor (IF). Hog IF can be replaced by human IF. However the important consideration is that IF preparations should be free of any possible contamination of other Cbl-binding proteins (nonIF). The supernatant fraction containing unbound IF-[57Co]Cbl and the pellet containing receptor bound IF-[57Co]Cbl are assayed for radioactivity. Receptor specific binding is calculated as the difference between the binding observed in the presence of CaCl2 and in the absence of calcium with EDTA. The most important step in the purification is the immunoaffinity chromatography using rabbit anti-hog intrinsic factor bound to Sepharose 4B. For the purification of the dog receptor attempts to use either intrinsic factor alone or the intrinsic factor-cobalamin complex as an affinity absorbent resulted in very poor binding of the receptor compared to rabbit anti-hog intrinsic factor antiserum. The advantage of this method is the recycling of both the intrinsic factor-cobalamin complex and the affinity ligand. After elution of the receptor the column can be washed with 0.2 M glycine-HCl buffer, pH 3.0, to remove the intrinsic factor-cobalamin complex which was bound to the antibody. The eluate is neutralized to pH 7 and can be reused after assessing the amount of complex present.

Original languageEnglish
Pages (from-to)23-28
Number of pages6
JournalMethods in enzymology
Volume123
Issue numberC
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

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