Investigation of the Peptide Chain of 124 kDa Phytochrome: Localization of Proteolytic Fragments and Epitopes for Monoclonal Antibodies

Abstract

Isolated oat phytochrome (124 kDa) was digested with endogenous proteases from oat and with trypsin. Fragments were isolated by SDS polyacrylamide gel electrophoresis (PAGE) blotted onto an activated glass fiber sheet and applied to microsequencing. Comparison of the amino terminal sequences of the fragments with the complete amino acid sequence derived from DNA sequence studies (Hershey et al., Nucleic Acid Res. 13, 8543 - 8560, [1985]) allowed the exact localization of each fragment. The reaction of the various fragments with monoclonal antibodies raised against 114/116 or 124 kDa oat or maize phytochrome were tested by means of Western blotting. Differential reactivity of the localized fragments allowed the localization of epitopes for several antibodies.