A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions
Spencer Schreier1,2, Bhanu Prakash Petla1, Tao Lin3
1Department of Agronomy, Horticulture, and Plant Science, South Dakota State University, Brookings, SD, 57007, USA.
Plant Molecular Biology
|October 18, 2019
Summary
A new, affordable assay quantifies DNA-protein interaction strength using tagged proteins and SYBR Gold stain. This method reveals distinct binding affinities of auxin response factor (ARF) proteins to DNA, impacting cellular auxin responses.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Quantifying DNA-protein interactions is crucial for understanding gene regulation.
- Existing methods can be expensive, complex, or require specialized reagents.
Purpose of the Study:
- To develop a simple, accessible, and inexpensive assay for quantifying DNA-protein interaction strength.
- To utilize this assay to compare the binding affinities of different auxin response factor (ARF) transcription factors to DNA targets.
Main Methods:
- Developed an assay using affinity resin to capture tagged recombinant proteins (e.g., 6xHis tag).
- Employed sequential washes and centrifugation to remove non-specific binding.
- Quantified captured DNA using SYBR Gold nucleic acid stain and fluorescence intensity.
Main Results:
- The assay accurately quantifies DNA-protein complex amounts, reflecting interaction strength.
- Demonstrated the assay's utility by comparing binding capacities of different ARF proteins to DNA targets, including AuxRE motifs.
- Observed distinct comparative affinities of ARF proteins for AuxRE variants.
Conclusions:
- The developed assay provides a versatile and quantitative method for assessing DNA-protein binding.
- Specific ARF-DNA binding strengths likely play a role in the intricate cellular auxin response.
- The assay's compatibility with standard laboratory equipment and reagents makes it broadly applicable.
