Immunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins.

Abstract

A method is described by which complex protein mixtures fractionated by standard 1-dimensional Na-DodSO4[sodium dodecyl sulfate]/polyacrylamide gel electrophoresis or O''Farrell 2-dimensional gel electrophoresis are then efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human [oral epidermoid carcinoma] KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins were detected by sequential treatment of diazobenzyloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 125I-labeled Staphylococcus aureus protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody. By 1-dimensional gel transfer/immunoautoradiography, 7 virion proteins were detected as prominent bands and several others as weaker bands. By 2-dimensional gel transfer/immunoautoradiography, several additional virus proteins were detected. Using anti-DNA binding protein serum, the MW 73,000 protein and MW 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.