Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• DNA-protein interactions are fundamental to cellular processes.
• Capillary electrophoresis (CE) offers rapid analysis with minimal sample consumption.
• Mobility shift assays are effective for studying these interactions.

Purpose of the Study:

• To investigate DNA-protein interactions using capillary electrophoresis (CE) with laser-induced fluorescence detection.
• To adapt mobility shift assay principles for CE-based analysis.
• To characterize the interaction between Escherichia coli trp repressor and its operator DNA.

Main Methods:

• Utilized capillary electrophoresis (CE) with laser-induced fluorescence detection.
• Employed fluorescently tagged operator DNA for detection.
• Optimized running buffer composition and pH for separation.
• Performed quantitative and qualitative analyses of repressor-operator complex formation.

Main Results:

• Demonstrated efficient separation of free operator DNA from the repressor-operator complex.
• Identified critical running buffer parameters (composition, pH) for optimal resolution.
• Quantified the formation of the repressor-operator complex by signal changes.
• Showcased selective separation of trp repressor from complex protein mixtures.

Conclusions:

• CE with laser fluorescence detection is a viable, non-radioactive method for studying DNA-protein interactions.
• Buffer conditions significantly impact the resolution and efficiency of separation.
• The assay can quantitatively and qualitatively assess repressor-operator binding specificity.