| Chapter title |
HTRF Kinase Assay Development and Methods in Inhibitor Characterization
|
|---|---|
| Chapter number | 1 |
| Book title |
Kinase Screening and Profiling
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3073-9_1 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3072-2, 978-1-4939-3073-9
|
| Authors |
Yong Jia, Mari Manuia, Jose Juarez |
| Abstract |
Due to their important roles in cellular signaling and their dysfunctions being linked to diseases, kinases have become a class of proteins being actively pursued as potential drug targets. Biochemical assays for kinases have been developed in various formats to facilitate inhibitor screening and selectivity profiling. Here, we focus on one such technology: homogeneous time-resolved fluorescence (HTRF). In this chapter, we describe the methods of developing an HTRF kinase assay using mutant EGFR enzyme as an example. We show how to determine the kinetic parameter of the enzyme (ATP K m), as well as how to study the inhibitor mechanism of action (MoA) exemplified by inhibitors of different MoAs. All methods described here can be readily applied to other kinases with minor modifications. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 8 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Unspecified | 1 | 13% |
| Student > Ph. D. Student | 1 | 13% |
| Student > Bachelor | 1 | 13% |
| Student > Master | 1 | 13% |
| Unknown | 4 | 50% |
| Readers by discipline | Count | As % |
|---|---|---|
| Unspecified | 1 | 13% |
| Agricultural and Biological Sciences | 1 | 13% |
| Medicine and Dentistry | 1 | 13% |
| Unknown | 5 | 63% |