Preparation, detection, and characterization of an antibody to rat alpha-phosphophoryn.
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 90 000, 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 alpha-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 alpha-phosphophoryn lacking the antigenic epitope of the antibody prepared.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003804:Dentin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007180:Incisor; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D000906:Antibodies; D000936:Antigen-Antibody Complex; D010750:Phosphoproteins; C017630:phosphophoryn