Chromophore-assisted laser inactivation of proteins by antibodies labeled with malachite green

Abstract

Chromophore-assisted laser inactivation (CALI) permits direct inactivation of proteins within cells or tissues with high spatial and temporal resolution. CALI converts nonfunction-blocking antibodies into function-blocking antibodies through the use of covalently linked chromophores, such as malachite green isothiocyanate (MGITC) and fluorescein isothiocyanate (FITC). Cells or tissues loaded with labeled antibody are exposed to laser light, which results in the generation of free radicals that locally damage the protein to which the antibody is attached. Protein function can be perturbed directly during precise periods of development without concerns accompanying the use of molecular genetic approaches, such as lethality or compensation. The dye resides in a cuvette in the dye laser module. Dyes are also available for fluorescein and GFP excitation wavelengths. The laser light is routed through the rear port of a Nikon Diaphot 200-inverted microscope or similar type of microscope. Since malachite green is strictly a chromophore, there is no fluorescence emission to observe during micro-CALI. However, the laser light itself is in the visible spectrum and may partially obscure the center of the image field.