Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.

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RESUMO

A better method was developed for analysis and identification of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins, bound to nitrocellulose, were identified by immunoblotting, while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography. The technique readily identified thyroid hormone receptors interacting with response elements representing inverted or direct repeats of the consensus half-site AGGTCA. In addition, specific antisera identified both the thyroid hormone and the retinoic acid receptors in heterodimeric complexes. Adding a third membrane and digoxigenin-labeled DNA probes allowed separate detection of [125I]T3 (labeled 3,5,3'-L-triiodothyronine), DNA, and protein from a single gel-shift reaction. The usefulness of this technique was also demonstrated by detecting the transcription factors P75gag-v-erbA and Jun in shifted complexes. Finally, proteins and DNA transferred to anion-exchange membranes can be eluted and subjected to further study. The combination of the gel shift and the immunoblot approaches (called "Shift-Western blotting") allows identification of the individual components of protein-DNA complexes containing multiple transcription factors, their cognate DNA elements and, when applicable, also the ligand.

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