Analysis of the interaction between A₁R and A_2AR proteins in living cells based on FRET imaging and batch processing method

Abstract

The quantitative FRET analysis of living cells is a tedious and time‐consuming task for freshman lacks technical training. In this study, FRET imaging and batch processing method were combined to analyze reagents‐induced interactions of A1R and A2AR on cell membranes. Results showed that the method had taken less time than if cell‐by‐cell was analyzed. The accuracy and repeatability of FRET efficiency values were likewise improved by removing the interference from anthropogenic factors. Then this method was applied to rapidly analyze acetaldehyde‐induced interactions, which analyzed hundreds of single‐cell trends by one operation, and the results revealed that interactions were consistently attenuated in LX‐2 cells, and statistical differences appeared after 30 min. Combined with batch processing method, procedures of cells FRET analysis have been greatly simplified without additional technical work, which has broad prospects in large‐scale analysis of cellar protein interaction.

Single-Nucleobase-Resolved Nanoruler Determines the Surface Energy Transfer Radius on the Living Cell Membrane

“…Distance-dependent energy transfer between a pair of a donor and an acceptor could be the most effective and practical optical nanoruler for sensing, imaging, and tracking biological and chemical events, such as protein–protein interactions, in vitro or in vivo imaging, protein conformational changes, , nucleic acid analysis, − binding site identification, , molecular tension measurements, etc . For example, Förster resonant energy transfer (FRET) proposed by Förster in 1948 opens up a new avenue for optical imaging − and tracking conformational changes of biological molecules, − as well as understanding deeper biochemical processes. − In FRET, two fluorophore molecular dipoles act as the donor and acceptor, which has a limited distance scale of less than 10 nm and greatly limits the detection of a broader range of molecular events.…”