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 journalArticlepeer-review

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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