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. 1985 Oct;82(20):6741–6744. doi: 10.1073/pnas.82.20.6741

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.

W K Miskimins, M P Roberts, A McClelland, F H Ruddle
PMCID: PMC390762  PMID: 2995982

Abstract

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.

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