Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry.
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.
Torrado
Mario
M
Institute of Health Sciences, University of La Coruña, La Coruña, Spain. torrado@udc.es
Iglesias
Raquel
R
Mikhailov
Alexander T
AT
D004926:Escherichia coli / Q000166:cytology; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013050:Spectrometry, Fluorescence / Q000379:methods*
D010446:Peptide Fragments; D049452:Green Fluorescent Proteins