Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.
Biosens Bioelectron
; 24(5): 1164-70, 2009 Jan 01.
Article
en En
| MEDLINE
| ID: mdl-18723336
In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Péptido Hidrolasas
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Trombina
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Técnicas Biosensibles
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Mapeo de Interacción de Proteínas
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Transferencia Resonante de Energía de Fluorescencia
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Mediciones Luminiscentes
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Revista:
Biosens Bioelectron
Asunto de la revista:
BIOTECNOLOGIA
Año:
2009
Tipo del documento:
Article
País de afiliación:
Australia
Pais de publicación:
Reino Unido