Southwestern blotting in investigating transcriptional regulation

Nature Protocols
Francis K Y SiuBilly K C Chow

Abstract

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...Continue Reading

References

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Citations

Apr 25, 2009·Journal of Proteome Research·Daifeng JiangHarry W Jarrett
Aug 11, 2011·Free Radical Biology & Medicine·M Hebert-SchusterV Nivet-Antoine
Jun 17, 2009·Nature Protocols·Antonio J PierikRoland Lill
Mar 9, 2012·Molecular and Cellular Biochemistry·Bipasha DeyVibha Rani
Jun 27, 2019·MBio·Jeffrey S KavanaughAlexander R Horswill
Sep 4, 2009·Journal of Chromatography. a·Daifeng JiangWilliam E Haskins

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