Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry
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Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometryData
2008Cita bibliográfica
Torrado M, Iglesias R, Mikhailov AT. Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry. Biotechniques. 2008;44(1):70-74
Resumo
[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.
Palabras chave
Escherichia coli
Genetic complementation test
Green fluorescent proteins
Peptide fragments
Microscopy-fluorescence
Protein binding
Protein interaction mapping
Spectrometry-fluorescence
Genetic complementation test
Green fluorescent proteins
Peptide fragments
Microscopy-fluorescence
Protein binding
Protein interaction mapping
Spectrometry-fluorescence
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ISSN
0736-6205
1940-9818
1940-9818