DNA-Binding Activity of Hypoxia-Inducible Factors (HIFs)
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Electrophoretic mobility shift assay (EMSA) provides a fast and sensitive method for detecting sequence-specific DNA-binding proteins ( see Fig. 1 ). Using an end-labeled DNA oligonucleotide as probe, proteins that specifically bind to these DNA sequence retard the mobility of the fragment during nondenaturing polyacrylamide gel electrophoresis (PAGE). This results in discrete band shifts corresponding to the individual protein-DNA complexes. This method can be used to investigate DNA-binding properties of purified proteins or of uncharacterized factors present in nuclear extracts. This assays also allows determination of affinity, sequence specificity, association and dissociation constants. Using specific antibodies that result in a supershift, individual proteins or protein complexes in DNA-binding activities can be identified. There is no single protocol that works equally for all proteins. We describe here a protocol designed to investigate DNA-binding properties of hypoxia-inducible factors (HIFs).
Fig. 1. Schematic drawing of electrophoretic mobility shift assays using a HIF-1 -binding site containing probe.
