Microarray analysis of protein-protein interactions based on FRET using subnanosecond-resolved fluorescence lifetime...

Area of Science:

• Biochemistry
• Biophysics
• Analytical Chemistry

Background:

• Protein-protein interactions are crucial in biological processes.
• Microarray-based assays are widely used for high-throughput screening.
• Traditional fluorescence intensity measurements can be susceptible to errors.

Purpose of the Study:

• To investigate the use of fluorescence lifetime as a dynamic parameter for detecting protein-protein binding on microarrays.
• To establish a Förster resonance energy transfer (FRET)-based competitive assay for protein interaction analysis.
• To evaluate the performance of this method across different microarray surfaces.

Main Methods:

• Utilized a FRET assay with biotinylated Bovine Serum Albumin (BSA) and streptavidin labeled with Alexa 555 (donor) and Alexa 647 (acceptor).
• Employed time-resolved fluorescence measurements with subnanosecond precision using a mode-locked laser and intensified CCD camera.
• Recorded fluorescence lifetime maps using the Rapid Lifetime Determination (RLD) scheme on various microarray surfaces.

Main Results:

• Successfully detected protein-protein interactions on CEL Epoxy, ARChip Epoxy, and ARChip Gel surfaces.
• Observed significant fluorescence quenching (up to 90%) upon streptavidin binding, indicating efficient FRET.
• Demonstrated good linearity of the quenching curves across all tested surfaces.

Conclusions:

• Fluorescence lifetime is a reliable and robust parameter for detecting protein-protein binding on microarrays.
• The FRET-based method provides a sensitive and accurate approach for analyzing protein interactions, overcoming limitations of fluorescence intensity.
• This technique is broadly applicable to various protein interaction studies on microarrays.