Rapid protein kinase assay using phosphocellulose-paper absorption

✍ Scribed by Jonathan J. Witt; Robert Roskoski Jr.

• Publisher: Elsevier Science
• Year: 1975
• Tongue: English
• Weight: 369 KB
• Volume: 66
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(75)90743-5

✦ Synopsis

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [y-"P]ATP as the phosphoryl donor. aliquots of the protein kinase assay mixture are applied to phosphocellulosepaper disks that are then immersed in water which elutes [y-3'P]ATP and metabolites. After brief organic solvent extraction and drying. bound radioactivity is measured by liquid scintillation spectrometry.