Capillary Electrophoresis with Laser-Induced Fluorescence Detection for the Analysis of Free and Immune-Complexed Green Fluorescent Protein

Abstract

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence (LIF). Exploiting recombinant technology and the natural fluorescence of GFP presents the capability of preempting the need for fluorescent derivatization. Such an approach would circumvent the obstacles typically associated with covalently labeling and purifying analytes that undergo fluorescent labeling. The unique property of GFP to fluoresce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly orin vitrowith wild-type GFP isoforms isolated from native jellyfishAequorea victoria.Second, GFP antisera were reacted with wild-type GFP and the formation of the GFP antigen–antibody complex was monitored. A simple borate buffer, pH 8.5, was ample for resolving the two isoforms of the naturally fluorescent GFP in less than 5 min. The separation of GFP from GFP–Ab was complete in less than 7 min with the individual components detectable at the picogram level. A number of factors influence CE separation and/or LIF detection including sample buffer pH and incorporation of the additive 1,4-diamino butane. Remarkably, conditions that severely impair fluorescence detection of free GFP do not diminish fluorescence detection of the GFP antigen–antibody complex in a similar manner. Thus, the antibody appears to preserve the natural fluorophore of GFP. These data lend credence to the utility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient separation and detection of an immunoreaction. In principle, a fusion protein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually required in LIF detection.