Assaying Protein Kinase Activity with Radiolabeled ATP

Abstract

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [γ-P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

“…Traditional methods measure tyrosine phosphorylation through the combination of radiolabeling with AT 32 P or AT 33 P, immunoprecipitation and western blotting. This strategy has been used to study tyrosine kinase activity for more than two decades with several protocol improvements made during these years (Karra, Stippec, & Cobb, 2017;Beeler, LaRochelle, Chedid, Tronick, & Aaronson, 1994;Saya et al, 1993). In a recent example of this approach, Marholz et al enabled assessing the catalytic activity of orphan PTKs (i.e.…”

An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation

“…The phosphorylation of 60 μM SrcKD kinase was achieved by His- and chitin-binding domain (CBD)-tagged Csk in the presence of 10 mM MgCl2, 10 mM DTT, 45 μM ATP, and 5% glycerol at room temperature for 30 minutes and was separated from His-CBD-CSK protein using Chitin beads. The phosphorylated SrcKD was successively tested for the extent of phosphorylation by 32 [P] assay 31 . The phosphorylated SrcKD was mixed with 0.5 molar excess of rPTPεD1 and the resultant complex was loaded onto a Superdex 200 HR 10/300 Increase column and separated using 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT buffer.…”

Activity‐Based Kinome Profiling Using Chemical Proteomics and ATP Acyl Phosphates

“…Traditional in vitro assays have been implemented to study kinase activity, including, e.g., radiochemical ATP assays (Hastie, McLauchlan, & Cohen, 2006;Karra, Stippec, & Cobb, 2017) and coupled bioluminescent assays (ADP-Glo; Tai, Bojjireddy, & Balla, 2011;Franks et al, 2017). Advantages of these assays include the potential for medium-to highthroughput analyses.…”