Torrado, MarioIglesias Reinoso, RaquelMikhailov, Alexander T.2018-05-152018-05-152008Torrado M, Iglesias R, Mikhailov AT. Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry. Biotechniques. 2008;44(1):70-740736-62051940-9818http://hdl.handle.net/2183/20705[Abstract] We have developed a set of simple modifications of the green fluorescent protein (GFP)fragment reassembly assay in bacteria that permits: (i)fluorescent microscopy visualization of GFP reassembly only 1-2 h after induction of protein expression, thus approximating the detection of GFP reassembly to the real-time dynamics of protein complex formation in living cells; (ii) spectrofluorometric detection of reassembled GFP fluorescent signals directly in lysates from cell suspension thereby avoiding, in many cases, the need for tag-affinity isolation of protein complexes; and (iii) comparative quantification of signal intensity in numerous cell-sample lysates using a Bio-Rad IQ5 spectrofluorometric detection system (Bio-Rad Laboratories, Madrid, Spain). Collectively, the results demonstrate that the combination of microscopic and spectrofluorometric detection provides a time-saving and sensitive alternative to existing methods of fluorescence complementation analysis.engEscherichia coliGenetic complementation testGreen fluorescent proteinsPeptide fragmentsMicroscopy-fluorescenceProtein bindingProtein interaction mappingSpectrometry-fluorescencejournal articleopen access