Radiometric Activity Assays

One of the biggest groups of proteins with evolutionary relationships is the protein kinase family. When treating different types of cancer, protein kinases are among the most successful therapeutic targets. The vastness of the protein kinase family has led to a great deal of research and development into screening technologies that work well for high-throughput screening (HTS) against broad panels of kinases and their mutations. Using radiometric activity assays, Creative Biolabs provides kinase drug screening services that screen compounds against extensive panels of kinases and their mutations.

Radiometric Filtration Binding Assay

The most common technique and industry standard for kinase profiling assays is the radioisotope filtration binding assay. It is the only format that does not require altered substrates or coupling enzymes to detect the real product directly. Test chemicals, such as ³²P-𝛾-ATP or ³³P-𝛾-ATP, are incubated with kinase, substrate, necessary cofactors, and radioisotope-labeled ATP in this experiment. The catalytic product labeled with radioisotopes is spotted onto p-81 phosphocellulose filter sheets after the reaction mixture has been incubated. The filter papers are then washed to eliminate any unreacted radioactive ATP. The main benefit of this approach is that it is a universal kinase test technique that works with any protein kinase without any restrictions. There is no need for any specific sub-stratum labeling or modification with this technology, and unreacted radioisotopes and fluorescent chemicals do not interfere with detection. Nevertheless, a significant barrier to using this technology for large-scale HTS is the requirement for radioisotopes as well as the filter cleaning and separation procedures.

Fig.1 The kinase assays in drug discovery. (Ma, et al, 2008) Fig.1. Kinase assay.¹

Applications of Radiometric Activity Assays

PAP³⁵S was used in conjunction with glycosaminoglycan substrates to measure the activity of chondroitin 6-ST (C6ST) and chondroitin 4-ST (C4ST). After enzyme conversion, the sulfated reaction products were separated by ethanol precipitation, membrane filtration, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of radioactivity incorporated into the polysaccharide was then determined by disaccharide analysis using HPLC and scintillation counting.
The activity of tyrosylprotein ST (TPST), which catalyzes the transfer of sulfo group from PAPS to the phenolic oxygen of tyrosine residues within highly acidic groups of proteins and polypeptides, was measured by measuring the transfer of ³⁵S sulfo groups to an immobilized peptide substrate using liquid scintillation counting. Using a medium-throughput radiolabel transfer-based assay, the well-characterized N-acetyl glucosamine (GlcNAc)-6-ST NodH from Rhizobium meliloti was examined.

The goal of kinase profiling is to ascertain a compound's specificity against a wide range of kinases. Because of this, a profiling assay format needs to be compatible with all of the kinases in the panel and unaffected by interferences from drugs and detections. Because it can be used to evaluate the functional activities of all protein kinases, the radioisotope-based filtration binding assay is one of the best options for kinase profiling activity tests. Please contact us for more information about our radiometric activity assays.

Reference

Ma, Haiching, Sean Deacon, and Kurumi Horiuchi. "The challenge of selecting protein kinase assays for lead discovery optimization." Expert opinion on drug discovery 3.6 (2008): 607-621.

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