Extraction and identification of electroimmunoprecipitated proteins from agarose gels.
A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.
Beyer
Natascha Helena
NH
Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark. nat@ssi.dk
Schou
Christian
C
Houen
Gunnar
G
Heegaard
Niels H H
NH
D000906:Antibodies; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D015415:Biomarkers / Q000032:analysis; D002021:Buffers; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D008970:Molecular Weight; D010449:Peptide Mapping; D013696:Temperature; D013997:Time Factors
D000906:Antibodies; D000941:Antigens; D015415:Biomarkers; D002021:Buffers