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Microscopy & Imaging JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/58763-v
What you'll learn
  • ✓ Perform automated fluorescence anisotropy measurements for transcription factor-DNA binding
  • ✓ Apply competitive titration assay methodology to determine binding affinities in solution
  • ✓ Interpret fluorescence microscopy data to quantify transcription factor-DNA interactions
  • ✓ Execute gel preparation and oligomer annealing for binding affinity experiments
Protocol

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.

Difficulty
advanced
Total time
~4–6 hours per sample (including gel preparation, annealing, image acquisition, and data fitting)

Steps

1
Anneal oligomer DNA for binding assay

Prepare and anneal complementary oligomer strands to generate double-stranded DNA templates for transcription factor binding.

▶ 01:00
2
Prepare gel matrix for microscopy

Cast and prepare polyacrylamide or agarose gel in a controlled delivery system suitable for fluorescence microscopy imaging.

▶ 01:45
3
Add competitor DNA and acquire images

Introduce competitor DNA solution to the gel matrix and perform automated image acquisition using fluorescence microscopy under controlled conditions.

▶ 03:35
4
Execute competitive titration and data fitting

Apply competitive titration assay protocol and use curve-fitting procedures to extract transcription factor-DNA binding affinity values from fluorescence anisotropy data.

▶ 04:41
5
Determine TF-DNA binding affinities from microscopy

Analyze fluorescence microscopy results to calculate and report quantitative transcription factor-DNA binding affinity measurements.

▶ 05:17
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