Back to Search
Start Over
Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
- Source :
- MethodsX, Vol 6, Iss , Pp 162-168 (2019)
- Publication Year :
- 2019
- Publisher :
- Elsevier, 2019.
-
Abstract
- Protein kinases play a crucial role in cellular functions by adding phosphate group to the protein substrates. It is an indispensable post-translational modification that regulates intracellular signaling and key cellular processes. They thus serve as an excellent target for chemotherapeutic interventions. A vast repertoire of protein kinases is present in a cell with diverse substrates as well as phosphorylation sites. To study full kinome for its activity, there is an urgent need of designing a comprehensive, in vitro assay which itself is an impractical task. However, in this study, we have attempted to develop a robust assay that not only mimics the in vivo nature of the kinases but can also be used in a high throughput drug-screening platform. Herein, the Leishmania donovani parasites are lysed and the total protein content is extracted. This extracted proteome is further sub divided into two parts: one active fraction containing cellular kinases and the substrate is heat-denatured fraction that loses all the enzymatic activity but retains the potential phosphorylation sites. These fractions are then co-incubated in the presence of ATP to initiate the kinase reaction and the total kinase activity is measured using ADP-glo kinase assay. Overall, this method • Presents a simple and robust approach to understand the participation of kinases in signaling networks. • Presents a high-throughput platform for ex-vivo drug screening. Method name: Kinome survey, Keywords: High throughput kinase assay, Leishmania donovani, Cell lysate, Non-radioactive
- Subjects :
- Science
Subjects
Details
- Language :
- English
- ISSN :
- 22150161
- Volume :
- 6
- Issue :
- 162-168
- Database :
- Directory of Open Access Journals
- Journal :
- MethodsX
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.63026b2b673140e5a9911b23003e5206
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.mex.2018.12.003