期刊
ANALYTICAL BIOCHEMISTRY
卷 424, 期 1, 页码 64-70出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.02.014
关键词
Palytoxin; Na,K-ATPase; Ouabain; Fluorescence polarization; Detection method
资金
- FEDER
- Ministerio de Ciencia y Tecnologia, Spain [AGL2007-60946/ALI, SAF2009-12581, AGL2009-13581-CO2-01, TRA2009-0189, AGL2010-17875]
- Xunta de Galicia, Spain [GRC 2010/10, PGIDT07CSA012261PR, PGDIT 07MMA006261PR, PGIDIT (INCITE) 09MMA003261PR, 2009/XA044, 2009/053, 2008/CP389, 10PX1B261254 PR]
- EU [211326-CP, 265896, 265409, 262649]
- Atlantic Area Programme (Interreg IVB Trans-national) [2008-1/003, 2009-1/117]
Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10 nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date. (c) 2012 Elsevier Inc. All rights reserved.
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