Journal of Cereal Science 34 (2001) 85–103 doi:10.1006/jcrs.2001.0380, available online http://www.idealibrary.com on Albumin and Globulin Proteins of Wheat Flour: Immunological and N-terminal Sequence Characterisation Jaswinder Singh*†‡, Malcolm Blundell*†, Greg Tanner† and John H. Skerritt*†§ *Quality Wheat Cooperative Research Centre Ltd and †CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia Received 12 September 2000 Abstract Several non-storage proteins are encoded by genes on individual wheat chromosomes and these have been used as genome-specific markers. In this study, a library of monoclonal antibodies with differing specificities to water- and salt-soluble proteins has been developed in order to obtain markers for different wheat chromosomes. Two antibodies, BCSAU 9D1 and JGM 1B4 were found to bind polypeptides encoded by chromosomes 3D and 4D respectively. Other antibodies bound to small numbers of polypeptides encoded by more than one chromosome, such as 5A, 7D, 5B and 5D. The proteins recognised by some of the antibodies were isolated by immunoaffinity chromatography, and one protein was identified as an alpha-amylase inhibitor by N-terminal sequence characterisation. In addition, 16 water-soluble and eight salt-soluble proteins were isolated using SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis and reversed-phase high performance liquid chromato- graphy, with the main objective being to confirm the identity of particular proteins, especially those which were able to be assigned to particular chromosomes. The N-terminal sequencing characterisation identified 19 proteins, whereas four proteins were found to be blocked and one did not match with any proteins in the database. Most of the water-soluble proteins belonged to a family of alpha-amylase inhibitors. One protein, assigned to chromosome 4BS, was homologous to serine carboxypeptidase III. N-terminal sequences of some of salt-soluble proteins matched with internal sequences of barley embryo globulin. Other proteins were identified as lipid transfer protein, peroxidase BP-1 precursor and histone H4 proteins. The protein sequences could also potentially be used for making antibody or DNA probes for use in selection in breeding programmes. 2001 Academic Press Keywords: monoclonal antibodies, N-terminal sequences, wheat grain, albumins, globulins. : BSA=bovine serum albumin; CHAPS=3-[(3-cholamidopropyl) dimethylammonio]-1 pro- pansulphonate; DTE=dithioerythreitol; ELISA=enzyme-linked immunosorbent assay; IEF=isoelectric focussing; IPG=immobilised pH gradient; MAb=monoclonal antibody; ME=2-mercaptoethanol; PBST=phosphate buffered saline containing Tween 20; RP-HPLC=reversed-phase high performance liquid chromatography; RT= room temperature; SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis. ‡ Present address: Research School of Biological Sciences, The Australian National University, GPO Box 475, Canberra, ACT 0200, Australia. E-mail: singh@rsbs.anu.edu.au § To whom correspondence should be addressed. Australian Centre for International Agricultural Research, GPO Box 1571, Canberra, ACT 2601, Australia. E-mail: skerritt@aciar.gov.au 0733–5210/01/040085+19 $35.00 2001 Academic Press