Protein kinase A
Problems in assay development often occur when the conditions required for sensitivity to the desired mechanism of action do not yield the best conditions for statistical reproducibility ; therefore, compromises and balances between these two opposing factors must be often made.
Li E, Hristova K. High Throughput Screen. Figure Table 2.
Protein Kinase Assay
To top. When designing protein kinase assays it is important first to consider the desired mechanism of action MOA of the inhibitor MOA is quite a complex topic which is nevertheless well described in terms of drug discovery strategies by David Swinney 27 ; examples are given of drugs that work by a variety of mechanisms and fall into three basic categories: Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins.
Longer incubation steps can add significant amounts of plate processing time in HTS automation because incubation time often represents the rate-limiting step in the HTS process. This allows for complex control of a pathway by means of multiple interacting kinases.
Growth factor receptor tyrosine kinases. Genome Biology. For instance, as one moves to smaller volumes, the surfaces available for nonspecific binding increase relative to the volume.
In this response, the hormone adrenaline causes the production of cAMP , a secondary messenger. Conventionally, the scintillation materials used in bioassays were liquids composed of aromatic hydrocarbons.
Protein kinase A - The School of Biomedical Sciences Wiki
Under normal circumstances, Src is predominantly inactive in cells, being switched on only at specific times. Stem cell factor mast cell growth factor receptor also known as the kit oncogene. Namespaces Page Discussion. Hunter T. Many substrates also have 'docking sites' that bind to the kinase outside of the active site, and many other kinases and substrates are brought together by scaffolding proteins or larger protein complexes, or by clustering in the plasma membrane.
Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: There are many commercially available kits and many published references describing these methodologies Table 1. The particular detector reagents to use, the assay reagent volumes, the concentrations of reagents, the incubation times, the buffer conditions Table 2 , the MTP types and the assay stopping reagents are all important parameters which need to be tested in order to obtain the very high level of reproducibility yet maintain the physiological and thermodynamic conditions to find a lead compound with the desired mechanism of action.
The advantages of using equal volumes are that it keeps the volumes at a level that is best for the particular pipette during automation; it minimizes the requirement for various specialized instruments and helps in the mixing of the reagents. Kinase assays that are coupled to luciferase have the disadvantage of being sensitive to luciferase inhibitors, which bind to the ATP-binding site of luciferase, resulting in false negatives. The problems encountered as one attempts to miniaturize an assay are in the change in surface to volume ratio, the lowered sensitivity and in the low volume dispensing of materials.
Protein tyrosine kinase inhibitors can act by binding directly in the ATP binding site competitively, type I inhibitors , but these tend to be less specific because of the shared characteristics of the ATP binding pocket among various kinases.
When cyclic AMP levels are low, catalytic subunits are bound to a regulatory subunit dimer and are inactive. As such, proteins like Csk and Chk are considered to have a tumour-suppressing ability.
The specific signal in the SPA is a consequence of a radiolabeled peptide or protein substrate becoming closely bound to the scintillation material. Proteins matched Defining balanced conditions for inhibitor screening assays that target bisubstrate enzymes. Within a given total assay volume, smaller volumes of reagents are added.