Identification of sequence-specific DNA-binding proteins by southwestern blotting.
We describe a Southwestern blotting method for characterization of both DNA-binding proteins and their specific sites. Proteins are first separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel, then renatured in SDS-free buffer and transferred by electroblotting to an immobilizing membrane, and detected by their ability to bind radiolabeled DNA. The protein(s) interacting with the labeled DNA is visualized by autoradiography. This technique was used in our laboratory to visualize the metal regulatory consensus sequence-binding protein MTF-1 in L cell crude nuclear extracts.
Centre de Recherche en Cancérologie de l'Université Laval, CHUQ, Pavillon l'Hôtel-Dieu de Québec, 11, côte de Palais, Québec, QC, G1R 2J6, Canada.
Simon
S
Labbé
Jean-François
JF
Harrisson
Carl
C
Séguin
D000818:Animals; D001483:Base Sequence; D026501:Blotting, Southwestern / Q000379:methods*; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000032:analysis*; D051379:Mice; D011485:Protein Binding; D014157:Transcription Factors / Q000032:analysis*; D000097809:Transcription Factor MTF-1
D002457:Cell Extracts; D004247:DNA; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1