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East Lothian, United Kingdom

Paterson M.J.,University of Dundee | Dunsmore C.J.,Elvingston Science Center | Hurteaux R.,Edinburgh Instruments | Maltman B.A.,Elvingston Science Center | And 2 more authors.
Analytical Biochemistry | Year: 2010

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17. ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the " gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements. © 2010 Elsevier Inc. Source

Thom J.,Elvingston Science Center | Anderson D.,Elvingston Science Center | McGregor J.,Elvingston Science Center | Cotton G.,Elvingston Science Center
Bioconjugate Chemistry | Year: 2011

Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins. © 2011 American Chemical Society. Source

Maltman B.A.,Elvingston Science Center | Dunsmore C.J.,Elvingston Science Center | Couturier S.C.M.,Almac science | Tirnaveanu A.E.,Elvingston Science Center | And 5 more authors.
Chemical Communications | Year: 2010

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured. © 2010 The Royal Society of Chemistry. Source

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