Abstract
Cytokines elicit responses in target cells by inducing changes in gene expression. For interferons (IFNs), this involves receptor-mediated activation of specific transcription factors, which then translocate into the nucleus to bind to cognate gene elements in the promoters of IFN-inducible genes. The prototypic IFN-inducible transcription factors are the signal transducer and activator of transcription (STAT) proteins. IFN-receptor interactions invoke Janus kinase activation via phosphorylation events, which in turn leads to the recruitment and phosphorylation of STAT proteins on tyrosine residues. Activated STATs then dimerize to form STAT complexes. IFNs-α/β will activate STAT-1, STAT-2, STAT-3 ,and STAT-5, whereas IFN-γ will predominantly activate STAT-1. In this chapter, we describe a procedure to identify IFN-inducible deoxyribonucleic acid (DNA) binding factors independently of any knowledge of their target DNA sequences. This procedure permits the identification of IFN-inducible STAT complexes as well as any other IFN-inducible DNA binding factors. This biochemical technique uses genomic DNA affinity chromatography to isolate DNA binding factors from IFN-inducible cytoplasmic or nuclear extracts.
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Add 30 µL of room temperature 5X SRB to the top of each membrane. Invert the membrane chambers into 1.5-mL Eppendorf tubes and centrifuge the tubes at ≥10,000g for 30 s at room temperature to collect the concentrated protein.
Acknowledgments
The authors acknowledge Dr. Julien Ghislain, who developed, tested, and established the GDAC protocol in this laboratory. He is currently based at the Ecole Normale Supérieure, Département de Biologie, Paris, France. We would like to thank M. Brierley for contributing the GDAC experiment data shown in Fig. 2 . The authors also thank B. Majchrzak-Kita and R. Deonarain for providing technical advice and for critical reading of this manuscript.
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Kumaran, J., Fish, E.N. (2005). Genomic DNA Affinity Chromatography. In: Carr, D.J.J. (eds) Interferon Methods and Protocols. Methods in Molecular Medicine™, vol 116. Humana Press. https://doi.org/10.1385/1-59259-939-7:057
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DOI: https://doi.org/10.1385/1-59259-939-7:057
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