Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper.
We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002854:Chromatography, Paper / Q000379:methods; D003979:Diazonium Compounds; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000939:Epitopes; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007668:Kidney; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D013539:Simian virus 40 / Q000032:analysis; D013205:Staphylococcal Protein A; D014764:Viral Proteins / Q000032:analysis / Q000276:immunology
D003979:Diazonium Compounds; D000939:Epitopes; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011506:Proteins; D013205:Staphylococcal Protein A; D014764:Viral Proteins; C030003:diazophenylthioether