A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli

Abstract

Protein synthesis initiation factor levels are measured in crude cell lysates of Escherichia coli MRE600 by use of a sensitive immunoblotting method. The method involves electrophoretic transfer of protein from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose paper and subsequent incubation with a specific antiserum and radioactive iodinated second antibody. The measurement of iodinated antibody attached to known amounts of initiation factor is determined by densitometric scanning of autoradiographs or counting radioactivity in excised protein bands. Linear standard curves over the range 1 to 300 ng of factor are obtained by these methods. Unknown amounts of initiation factor in crude cell lysates are measured accurately; values agree with previous radioimmune assay data. The immunoblotting method serves as an alternative to the radioimmune assay in measuring small quantities of protein in complex mixtures. Immunoblotting enjoys three major advantages: it is simple and rapid to execute; it is sensitive; and it is capable of distinguishing multiple forms of the antigen which separate in the gel system employed.