A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

Christina Spry; Kevin J. Saliba; Erick Strauss

doi:10.1016/j.ab.2013.12.010

A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

Christina Spry^*, Kevin J. Saliba, Erick Strauss

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

12 Citations (Scopus)

Abstract

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)₂ and ZnSO₄ are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

Original language	English
Pages (from-to)	76-78
Number of pages	3
Journal	Analytical Biochemistry
Volume	451
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2013.12.010
Publication status	Published - 15 Apr 2014

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title = "A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices",

abstract = "We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.",

keywords = "Choline kinase, Hexokinase, Kinase, Pantothenate kinase, Phosphorylation assay, Somogyi",

author = "Christina Spry and Saliba, {Kevin J.} and Erick Strauss",

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T1 - A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

AU - Spry, Christina

AU - Saliba, Kevin J.

AU - Strauss, Erick

PY - 2014/4/15

Y1 - 2014/4/15

N2 - We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

AB - We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

KW - Choline kinase

KW - Hexokinase

KW - Kinase

KW - Pantothenate kinase

KW - Phosphorylation assay

KW - Somogyi

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DO - 10.1016/j.ab.2013.12.010

M3 - Article

SN - 0003-2697

VL - 451

SP - 76

EP - 78

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -

A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

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