CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION FOR THE ANALYSIS OF FREE AND IMMUNE-COMPLEXED GREEN FLUORESCENT PROTEIN

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluoresc ence (LIF). Exploiting recombinant technology and the natural fluoresc ence of GFP presents the capability of preempting the need for fluores cent derivatization. Such an approach would circumvent the obstacles t ypically associated with covalently labeling and purifying analytes th at undergo fluorescent labeling. The unique property of GFP to fluores ce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly or in vitro with wild-type GFP isoforms isolated from n ative jellyfish Aequorea victoria. Second, GFP antisera were reacted w ith wild-type GFP and the formation of the GFP antigen-antibody comple x was monitored. A simple berate buffer, pH 8.5, was ample for resolvi ng the two isoforms of the naturally fluorescent GFP in less than 5 mi n. The separation of GFP from GFP-Ab was complete in less than 7 min w ith the individual components detectable at the picogram level. A numb er of factors influence CE separation and/or LIF detection including s ample 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-ant ibody complex in a similar manner. Thus, the antibody appears to prese rve the natural fluorophore of GFP. These data lend credence to the ut ility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient sepa ration and detection of an immunoreaction. In principle, a fusion prot ein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually requ ired in LIF detection. (C) 1997 Academic Press.