Journal
/
Biochemistry
/
High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
JoVE Journal
Biochemistry
This content is Free Access.
JoVE Journal Biochemistry
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

•

06:38 min

•

February 07, 2019

•

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

Chapters

  • 00:04Title
  • 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

Summary

Automatic Translation

Automatic Translation

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.

Tags

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

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

check_url/58763?article_type=v

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
check_url/58763?article_type=v

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
check_url/58763?article_type=v

Measuring Protein Binding to F-actin by Co-sedimentation
check_url/58763?article_type=v

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms
check_url/58763?article_type=v

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging
check_url/58763?article_type=v

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
check_url/58763?article_type=v

Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy
check_url/58763?article_type=v

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy
check_url/58763?article_type=v

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling
check_url/58763?article_type=v

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Read Article