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Southwestern blotting in investigating transcriptional regulation

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 32P-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 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.

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Figure 1: A flow chart summarizing the approximate time needed and the procedures involved in Southwestern blotting.
Figure 2: A flow chart showing a strategy for identifying DNA-binding proteins (e.g., transcription factors) by Southwestern blotting in combination with other methods.
Figure 3: Southwestern blotting of the N1 motif with SH-SY5Y nuclear extracts.

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Acknowledgements

The work was supported by Hong Kong Government RGC Grants HKU7501/05M, HKU7639/07M to B.K.C.C.

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F.K.Y.S. and L.T.O.L. contributed equally to this work, and should be considered as cofirst authors.

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Correspondence to Billy K C Chow.

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Siu, F., Lee, L. & Chow, B. Southwestern blotting in investigating transcriptional regulation. Nat Protoc 3, 51–58 (2008). https://doi.org/10.1038/nprot.2007.492

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