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 medium 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.
D000941:Antigens / Q000032:analysis* / Q000276:immunology; D002241:Carbohydrates / Q000276:immunology; D002473:Cell Wall / Q000032:analysis / Q000276:immunology; D002696:Chlamydomonas / Q000276:immunology*; D003429:Cross Reactions; D003636:DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005407:Flagella / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology; D008970:Molecular Weight; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*
D000941:Antigens; D002241:Carbohydrates; D000939:Epitopes; D006023:Glycoproteins; D003636:DEAE-Cellulose