Reference "Separation and characterization of basic barley seed proteins"

Reference ID

8629

Title

Separation and characterization of basic barley seed proteins

Source

Electrophoresis, 2000, vol. 21, pp. 3693-3700

Authors (2)

Kristoffersen-H-E

Flengsrud-R

Abstract

Basic proteins in barley starchy endosperm from developing seeds were separated
by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as
partial extracts were analyzed. Edman degradation sequencing and immunological
detection were performed after transfer of separated proteins onto membranes.
Only one protein could be analyzed by N-terminal sequencing of blotted and
separated proteins from the total extract. Fractionation of extracts was done
using cation exchange chromatography, concanavalin A and heparin affinity
chromatography. Internal sequences were determined after in-gel cleavage of
proteins using trypsin or cyanogen bromide and separation of the fragments by
reversed-phase chromatography or in a gel electrophoresis system for peptide
separation. This resulted in a new protocol for obtaining internal sequences
from proteins separated by 2-D electrophoresis. A total of 16 sequences,
including nine internal sequences, were analyzed, permitting the identification
of ten proteins, including five that appeared to have a blocked N-terminus. An
additional protein was identified using immunological detection. Three protein
sequences remained unidentified. Separated proteins were also analyzed with a
glycan detection method.