Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.
We describe a procedure for detecting high-affinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005796:Genes; D006801:Humans; D009698:Nucleoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D011990:Receptors, Transferrin; D012208:Rhabdomyosarcoma; D014168:Transferrin / Q000378:metabolism
D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D004247:DNA