Affinity assays using fluorescence anisotropy with capillary electrophoresis separation

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biophysics

Background:

• Affinity interactions are crucial for biological processes.
• Accurate detection and quantification of these interactions are essential for drug discovery and diagnostics.
• Existing methods may lack the sensitivity or specificity required for complex biological samples.

Purpose of the Study:

• To develop a novel method combining fluorescence anisotropy and capillary electrophoresis (FACE) for detecting molecular binding.
• To provide a technique that offers both separation and confirmation of binding events.
• To establish a sensitive and quantifiable assay for identifying binding partners.

Main Methods:

• Developed a Fluorescence Anisotropy with Capillary Electrophoresis (FACE) assay.
• Utilized a fluorescent probe with low fluorescence anisotropy.
• Detected binding through a positive anisotropy shift during capillary electrophoresis.
• Employed calculations combining the Perrin equation and dissociation constant to predict assay performance.

Main Results:

• Experimental results aligned with theoretical predictions regarding probe and protein concentrations.
• Demonstrated successful detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe.
• Achieved a limit of detection of 3 nM for G(alphai1) with 250 nM probe concentration.

Conclusions:

• The FACE assay effectively detects and quantifies molecular binding interactions.
• The method offers high sensitivity, with a low limit of detection for specific binding partners.
• FACE has potential applications in screening complex samples for compounds with affinity for target molecules.