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.
authors: Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia. Electronic address: christina.spry@anu.edu.au.
authors: Christina
authors: C
authors: Spry
authors: Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia; Medical School, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia.
authors: Kevin J
authors: KJ
authors: Saliba
authors: Department of Biochemistry, Stellenbosch University, Matieland 7602, South Africa. Electronic address: estrauss@sun.ac.za.
authors: Erick
authors: E
authors: Strauss
mesh_terms: D017609:Barium Compounds / Q000737:chemistry; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D008904:Miniaturization; D010205:Pantothenic Acid / Q000737:chemistry / Q000378:metabolism; D010758:Phosphorus / Q000032:analysis* / Q000737:chemistry; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011874:Radiometry; D019287:Zinc Sulfate / Q000737:chemistry
chemical_list: D017609:Barium Compounds; D029968:Escherichia coli Proteins; D010761:Phosphorus Radioisotopes; D010205:Pantothenic Acid; C012766:barium hydroxide; D010758:Phosphorus; D019287:Zinc Sulfate; D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase
keywords: Choline kinase; Hexokinase; Kinase; Pantothenate kinase; Phosphorylation assay; Somogyi