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Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.
- Source :
-
Talanta [Talanta] 2016 Aug 15; Vol. 156-157, pp. 1-5. Date of Electronic Publication: 2016 Apr 27. - Publication Year :
- 2016
-
Abstract
- We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).<br /> (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Subjects :
- Cyclic AMP-Dependent Protein Kinases analysis
Halogenation
Indicators and Reagents
Oligopeptides analysis
Oligopeptides isolation & purification
Phosphopeptides analysis
Phosphopeptides isolation & purification
Phosphorylation
Rhodamines analysis
Rhodamines isolation & purification
Rhodamines metabolism
Spectrometry, Fluorescence methods
Cyclic AMP-Dependent Protein Kinases metabolism
Enzyme Assays methods
Ferric Compounds chemistry
Imino Acids chemistry
Oligopeptides metabolism
Phosphopeptides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1873-3573
- Volume :
- 156-157
- Database :
- MEDLINE
- Journal :
- Talanta
- Publication Type :
- Academic Journal
- Accession number :
- 27260427
- Full Text :
- https://doi.org/10.1016/j.talanta.2016.04.058