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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
An efficient and cost-effective protocol for selecting transcription factor binding sites that reduces isotope usage
Joseph Krystel1, Kasirajan Ayyanathan
1Center for Molecular Biology and Biotechnology, Florida Atlantic University, Jupiter, Florida 33458, USA.
Journal of Biomolecular Techniques : JBT
|September 7, 2012
Summary
This study presents a modified binding site selection (BSS) protocol that reduces radioisotope use for identifying transcription factor DNA binding sites. The new method enhances efficiency and maintains assay efficacy, aiding in understanding transcription factor activity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Transcription factors regulate gene expression by binding DNA in a sequence-specific manner.
- Identifying these DNA binding sites is crucial for understanding transcription factor activity.
- Standard methods often rely on radioisotopes or expensive equipment, limiting accessibility and efficiency.
Purpose of the Study:
- To develop a modified binding site selection (BSS) protocol that improves efficiency and reduces radioisotope usage.
- To pre-enrich DNA-binding libraries for transcription factors, streamlining subsequent selection processes.
- To maintain the efficacy of binding site selection while decreasing reliance on radioactive materials.
Main Methods:
- A modified BSS protocol was developed using a GST affinity-tagged DNA binding domain immobilized on a GSH affinity column.
- A randomized oligonucleotide library was selected using the immobilized protein.
- The pre-enriched library was then used in standard electrophoretic mobility shift assay (EMSA) selection rounds.
- The protocol was applied to identify binding sequences for Drosophila melanogaster transcription factors.
Main Results:
- The modified BSS protocol successfully pre-enriched oligonucleotide libraries for transcription factor binding.
- Using the pre-enriched library significantly reduced the number of labeled rounds required in EMSA.
- The protocol decreased radioisotope usage while maintaining the efficacy of transcription factor binding site selection.
- Consensus binding sequences were identified for several Drosophila transcription factors.
Conclusions:
- The developed modified BSS protocol offers a more efficient and less radioisotope-dependent method for identifying transcription factor DNA binding sites.
- This approach facilitates the study of transcription factor activity and DNA-protein interactions.
- The protocol is effective for various transcription factors, including those from Drosophila melanogaster.
