Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.
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.
Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
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S
Demczuk
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M
Harbers
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Vennström
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone / Q000032:analysis / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D000906:Antibodies; D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D014157:Transcription Factors; D014284:Triiodothyronine; D014212:Tretinoin; D004247:DNA