ELECTROTRANSFER OF SDS-PAGE SEPARATED POLYPEPTIDES TO THE DE81 BLOTTING MATRIX AND DETECTION OF CHLAMYDOMONAS ANTIGENS AND GLYCOCONJUGATES

Electrotransfer of SDS-PAGE-separated polypeptides to nitrocellulose is not quantitative under the conditions described by Towbin et al. (1979). The use of FITC-labelled polypeptide markers and FITC-labelled Chlamydomonas flagella has allowed investigation of separate aspects of the electrotransfer process. These aspects include electroelution from the polyacrylamide gel, binding to the blotting matrix and electrophoretic re-elution from the blotting matrix. Factors which influence electrotransfer, including electrophoretic field strength, time-dependence of electrotransfer, the effect of mediuum composition and the binding capacity of the DE81 blotting matrix have been examined. SDS-PAGE-separated polypeptides up to Mr 350,000 can be electrotransferred to DE81 in a nearly quantitative manner in a dilute Laemmli (1970) electrophoresis medium containing 0.05% SDS in an electric field of 4 V/cm for 4 h. The efficient electrotransfer of polypeptides over a wide Mr range has allowed a study of the cross-reactivity of polyclonal antisera raised against Chlamydomonas cell walls, isolated flagella and the flagellar 350,000 major membrane glycoprotein. The principal epitopes recognised by the cross-reactive antibodies appear to be periodate-sensitive carbohydrate residues of cell wall and flagellar glycoproteins. These epitopes do not appear to include ConA binding sites.