Chromophore-assisted laser inactivation.
The major challenge of the post-genome world is ascribing in situ function to the myriad of proteins expressed in the proteome. This challenge is met by an arsenal of inactivation strategies that include RNAi and genetic knockout. These are powerful approaches but are indirect with respect to protein function and are subject to time delays before onset and possible genetic compensation. This chapter describes two protein-based inactivation approaches called chromophore-assisted laser inactivation (CALI) and fluorophore-assisted light inactivation (FALI). For CALI and FALI, light inactivation is targeted via photosensitizers that are localized to proteins of interest through antibody binding or expressed domains that are fluorescent or bind fluorescent probes. Inactivation occurs when and where the cells or tissues are irradiated and thus CALI and FALI provide an unprecedented level of spatial and temporal resolution of protein inactivation. Here we provide methods for the labeling of antibodies and setup of light sources and discuss controls, advantages of the technology, and potential pitfalls. We conclude with a discussion on a number of new technologies derived from CALI that combine molecular genetic approaches with light-induced inactivation that provide new tools to address in situ protein function.
Hoffman-Kim
Diane
D
Department of Molecular Pharmacology, Physiology, and Biotechnology, Center for Biomedical Engineering, Brown University, Providence, Rhode Island 02912, USA.
Diefenbach
Thomas J
TJ
Eustace
Brenda K
BK
Jay
Daniel G
DG
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D007834:Lasers
D000906:Antibodies; D016650:Fluorescein-5-isothiocyanate