Cold Spring Harbor Protocols vol:2011 pages:pdb.prot5596
INTRODUCTION Fluorescein-assisted light inactivation (FALI) is a powerful method for studying acute loss of protein function, even if the corresponding mutations lead to early lethality. In this protocol, FALI is mediated by the membrane-permeable FlAsH (4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein) compound that binds with high specificity to the genetically encoded tetracysteine tag and thus allows the inactivation of protein function in vivo with exquisite spatial (<40 Å) and temporal (<30 sec) resolution. It also enables the analysis of kinetically distinct processes such as synaptic vesicle exocytosis and endocytosis. This protocol describes the creation of a tetracysteine-tagged construct that can be used in FlAsH-FALI. These tagged genomic constructs are cloned in a conditional amplifiable bacterial artificial chromosome (BAC), such as P(acman), using recombineering.