Understanding gene regulation by Hox transcription factors requires understanding the forces that underlie DNA binding by these proteins. Electrophoretic mobility shift analysis (EMSA) not only allows measurement of protein affinity and cooperativity but also permits visualization of differently migrating protein-DNA complexes, including complexes with different compositions or complexes with identical compositions yet assembled in different geometries. Furthermore, protein activity can be measured, allowing correction of binding constants for the percentage of protein that is properly folded and capable of binding DNA. Protocols for measuring protein activity and the equilibrium DNA-binding dissociation constant (Kd) are provided. This versatile assay system can be adjusted based on specific needs to measure other parameters, including the kinetic association and dissociation constants (ka and kd) and the formation of heterologous protein-protein interactions.
Hox Gel retardation Gel shift Electrophoretic mobility shift analysis EMSA DNA binding Activity Affinity Cooperativity
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This work was supported by an RDEAP grant from the Texas A&M Health Science Center to S.E.B. and a Robert A. Welch Foundation grant (C-576) to K.S.M.
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