Southwestern blotting in investigating transcriptional regulation

• Published in: Nature protocols Vol. 3; no. 1; pp. 51 - 58
• Main Authors: Chow, Billy K C, Siu, Francis K Y, Lee, Leo T O
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.01.2008
• Subjects: Autoradiography; Blotting, Southwestern - methods; Cell Line; Database searching; Deoxyribonucleic acid; DNA; DNA binding proteins; DNA Probes; DNA-Binding Proteins - analysis; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Genetic transcription; Humans; Membranes; Molecular weight; Nucleic Acid Hybridization; Physiological aspects; Proteins; Protocol; Transcription factors; Transcription Factors - analysis; China
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2007.492

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.