.png)
In vitro kinase assays of ABL tyrosine kinase.
Abltide-GST was used as a substrate.
ATP or ATP-γS was used as a phosphate or thiophosphate doner.
ATP-γS has the advantage of permitting measurements of kinase activities without using radioisotope, and thiophosphorylated proteins were detected sensitively by using excellent antibody. However, the question remains whether particular kinases accept
ATP-γS as readily as they accept natural ATP.
The Phos-tag gel permitted analysis of thiophosphorylated proteins as well as phosphorylated proteins and it identified differences in the efficiencies of utilization of ATP-γS and ATP
in these reactions.
To determine absolute quantities of the kinase reaction products of Abltide-GST, we used densitometric analysis to calculate the ratio of the thiophosphorylated or phosphorylated
form to the total substrate in each lane of the Zn2+––Phos-tag SDS-PAGE gel.
This shows that the utilization efficiency of ATP-γS is much less than that of ATP.
Related data
Glivec (ABL inhibitor) inhibition profile
Abl/Abltide-GST kinase assay
Abl/Abltide-GST phosphatase assay