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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
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Biyokimya
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JoVE Journal Biyokimya
High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

DOI:
10.3791/58763-v

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06:38 min

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February 07, 2019

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, , , ,
1Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM),Ludwig-Maximilians-Universität München

Bölümler

  • 00:04Başlık
  • 01:00Oligomer Annealing
  • 01:45Gel Preparation
  • 03:35Addition of the Competitor DNA Solution and Image Acquisition
  • 04:41Competitive Titration Assay and the Fitting Procedure
  • 05:17Results: Determining TF-DNA Binding Affinities Using Fluorescence Microscopy
  • 06:07Conclusion

Özet

Otomatik Çeviri

Otomatik Çeviri

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Etiketler

Transcription Factor-DNA BindingFluorescence MicroscopyDissociation ConstantCompetitive TitrationAnisotropy MeasurementBinding AffinityProtein-DNA InteractionDNA AnnealingAgarose GelTitration PlateCalibration WellNile Blue Dye

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