A High-Throughput Radiometric Kinase Assay

Abstract

Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases.

The Expression Pattern of the Phosphoproteome Is Significantly Changed During the Growth Phases of Recombinant CHO Cell Culture

“…(See Table S5, Supporting Information for the top 20 most‐enriched GO: Biological Processes and GO: Molecular Functions from the phosphoproteomic and the non‐enriched whole cell lysate proteomic analyses). We also identified the differential expression of 94 kinases along the growth curve, as shown in Table S6, Supporting Information, which is a remarkable number given that the human kinome contains close to 518 kinases …”

Kinase Sensing Based on Protein Interactions at the Catalytic Site

“…In addition, while many biosensors require multistep assembly, our biosensor, only requires the simple and standard thiol‐mediated adsorption on gold surfaces in a single step, making it highly versatile. Both previous and current kinase sensing methods require the use of radio‐labelled ATP [8] . This derivative is expensive, requires immediate use and raises unnecessary health risks.…”

“…Accordingly, abnormal kinase activity is closely connected to many diseases, including various types of cancer [2] and kinases are considered to be promising cancer biomarkers [3,4] . Many analytical methods have been developed for kinase activity quantification as prognostic tools, [5,6] including radiometric, [7,8] spectroscopic [9–11] and immunological tools [12,13] . Electrochemical methods were also developed for kinase detection, [14–18] and as all electrochemical tools they have the distinct advantages of relatively low cost, easy operation, rapid response and high sensitivity [19] .…”

Identification and characterization of the Cucurbitacins, a novel class of small-molecule inhibitors of Tropomyosin receptor kinase a

“…Aberrant kinase function or regulation can contribute to the rise of many diseases [20]. While protein kinases have become therapy targets, only a small fraction of protein kinases are targeted by validated inhibitors [21]. High-throughput screening technology has become a key tool to screen several compounds against kinases quickly and effectively [21, 22].…”

“…While protein kinases have become therapy targets, only a small fraction of protein kinases are targeted by validated inhibitors [21]. High-throughput screening technology has become a key tool to screen several compounds against kinases quickly and effectively [21, 22]. In this study, we used kinase screening approaches to identify kinase targets of CuB.…”