Preparation, Detection, and Characterization of an Antibody to Rat α-Phosphophoryn

Tzong Guang Tsay, Arthur Veis*

*Corresponding author for this work

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

The phosphophoryn components of rat incisor dentin were extracted under stringent conditions to prevent proteolytic degradation during processing. Successive steps of CaCl2 precipitation, ion-exchange chromatography, and gel filtration over Biosil TSK G4000SW high-performance liquid chromatography columns in 4.0 M guanidine hydrochloride yielded a mixture of phosphoryns that contained only trace amounts of other proteins, as shown by a very sensitive double stain procedure on acrylamide gels after electrophoresis. The highest weight phosphophoryn had a molecular weight of 90000, on gradient polyacrylamide gels calibrated with globular protein standards. Polyclonal antibodies were produced in rabbits following a complex scheme. The specific antibodies were collected by passage over a Sepharose column conjugated to the purified phosphophoryn. The isolated antibody was used to prepare a second affinity column. Passage of the initial phosphophoryn fraction over the column led to the retention of a single component, identified as the Mr 90 000 α-phosphophoryn. Thus, a monospecific polyclonal antibody has been prepared. These data show that the other phosphoryns of the rat incisor must be distinct species or slightly degraded products of the a-phosphophoryn lacking the antigenic epitope of the antibody prepared.

Original languageEnglish (US)
Pages (from-to)6363-6369
Number of pages7
JournalBiochemistry
Volume24
Issue number23
DOIs
StatePublished - Nov 1 1985

ASJC Scopus subject areas

  • Biochemistry

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