Summary
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.
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This work was supported by a Grant from the Conseil de recherches en sciences naturelles et en génie du Canada to CS.
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Labbé, S., Harrisson, JF., Séguin, C. (2009). Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_12
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DOI: https://doi.org/10.1007/978-1-60327-015-1_12
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