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PMID: 2995982 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 82 ·No. 20 ·1985-10-00 ·Pages 6741-4

Miskimins WK, Roberts MP, McClelland A, Ruddle FH

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.

MeSH Terms
Base Sequence Cell Line DNA/metabolism DNA-Binding Proteins/metabolism Genes Humans Nucleoproteins/genetics Promoter Regions, Genetic Receptors, Cell Surface/genetics,metabolism Receptors, Transferrin Rhabdomyosarcoma Transferrin/metabolism
Chemicals
DNA-Binding Proteins Nucleoproteins Receptors, Cell Surface Receptors, Transferrin Transferrin DNA
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Miskimins W K
Roberts M P
McClelland A
Ruddle F H
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31 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.82.20.6741
Published
1985-10-00
Pages
6741-4
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC390762
Subset
IM
Grants
NIGMS NIH HHS · GM09966 · United States
External Links
PubMed source DOI: 10.1073/pnas.82.20.6741 PMC Full Text View journal details
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