A High-Throughput Radiometric Kinase Assay.
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 of 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.
Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA.
Krisna C
KC
Duong-Ly
Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA. jeffrey.peterson@fccc.edu.
Jeffrey R
JR
Peterson
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007202:Indicators and Reagents; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D011874:Radiometry / Q000379:methods*; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors; D054852:Small Molecule Libraries / Q000494:pharmacology
D007202:Indicators and Reagents; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D000255:Adenosine Triphosphate; D011972:Receptor, Insulin
Dot blot kinase assay; High-throughput screen; Insulin receptor; Kinase; Kinase assay; Kinase inhibitor