An indirect-labeling procedure for the study of specific interactions of proteins with DNA

A semiquantitative and reproducible indirect-labeling procedure for the study of specific protein/DNA interactions using nitrocellulose-filter-immobilized proteins and linear or superhelical DNA molecules is reported. Proteins were immobilized on nitrocellulose filters either by direct dotting or by electrotransfer from polyacrylamide electrophoretic gels. After incubation with the respective DNA (linear restriction fragments or closed circular recombinant DNA plasmids) the paper strips were washed, and specifically bound DNA was denatured by alkali and detected by hybridization with 32P-nick-translated DNA. The quantitation of the reaction was performed by scanning of the autoradiograms of the radioactive spots and determination of the area under the respective peaks. The intensity of the radioactive spots was proportional to the amount of protein present in the dot. The sensitivity of the assay depends primarily on the affinity of the respective DNA to the protein and in the case of mouse liver histone H1AB/mouse alpha-globin gene approximately 50 ng of protein per dot was enough for determination.