29] Fluorophore-assisted light inactivation for multiplex analysis of protein function in cellular processes

Abstract

This chapter describes the fluorophore-assisted light inactivation (FALI) for multiplex analysis of protein function in cellular processes. The application of photons to the imaging and analysis of biological processes has made great strides. This includes the ability to observe single-molecule interactions and advances in light microscopy using green fluorescent protein (GFP) fusions and fluorescence energy transfer to show dynamic protein interactions in cells. Others have extended this approach by developing chromophore-assisted laser inactivation (CALI). CALI is a method to disrupt protein function in cellular context as a means to address the roles that specific proteins play in cells and model organisms. CALI is an effective technique to directly address protein function in cells and a high-throughput CALI approach would be beneficial for multiple applications. To develop high-throughput applications, the chapter investigates an alternative approach to inactivate proteins in cells using diffuse light. FALI allows applying light-mediated protein inactivation to samples in a multiplex fashion to address protein function in cellular processes with potentially high throughput.