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Binding Reaction

Nucleic acid probes

Short linear DNA fragments that contain consensus binding sequences are typically used as probes.

  • Existing EMSA protocols are easily converted to NIR fluorescent assays by replacing the existing DNA oligonucleotides with IRDye end-labeled oligonucleotides.
  • Binding and electrophoresis conditions are very similar to other EMSA detection methods.
  • After electrophoresis, fluorescent protein:DNA complexes can be imaged immediately while still in the gel, without gel drying or transfer.1-4

Using competitor DNAs

  • Non-specific competitors [irrelevant, unlabeled nucleic acids such as poly(dI-dC) or salmon sperm DNA] are used as blocking agents, to decrease binding of non-specific proteins to the labeled DNA probe.
  • Specific competitors are important controls used to confirm specificity of protein:DNA binding. Typically, the specific competitor contains exactly the same consensus sequence as the labeled probe. The competitor is unlabeled, and is added to the binding reaction in a large molar excess (~200-fold).
    • Unlabeled competitor DNA out-competes the labeled probe for binding to the protein, eliminating or reducing the mobility shift.
    • Mutant competitors can also be used. They contain mutated or low-affinity binding sites that will not compete with specific interactions and do not reduce the observed mobility shift.
  • 1. Shaik, SS et al. J Biol Chem 284(9): 5945-55 (2009)
  • 2. Zhang, S et al. Proc Natl Acad Sci USA 105(11): 4156-61 (2008)
  • 3. Berg, DT et al. J Biol Chem 280(15): 14943-7 (2005)
  • 4. Jullien, N and J-P Herman. BioTechniques 51: 267-269 (2011)

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