SEB Supplemented Enzymatic Buffer

The Supplemented Enzymatic Buffer (SEB) is a proprietary substrate stabilizing component that may be used with KinEASE TK assay to optimize the enzymatic reaction for some tyrosine kinases while maintaining inhibitor properties such as IC50.
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The Supplemented Enzymatic Buffer (SEB) is a proprietary substrate stabilizing component that may be used with KinEASE TK assay to optimize the enzymatic reaction for some tyrosine kinases while maintaining inhibitor properties such as IC50.
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Overview

Some Tyrosine kinase enzymatic activity may require further assay optimization for a sensitive detection of phosphorylation levels. The Supplemented Enzymatic Buffer is a substrate stabilizing buffer that may be used to achieve an optimal enzymatic step prior to detection with the HTRF KinEASE TK kit, while maintaining inhibiting compound pharmacological characteristics.

Supplemented Enzymatic Buffer performance

The HTRF KinEASE TK reaction may be run in the presence of SEB, a proprietary substrate stabilizing buffer. This buffer greatly enhances S/B and enables the use of lower kinase concentrations per well. Among validation on other tyrosine kinases, the case below shows the impact of SEB on tests conducted with Abl (Merck Millipore 14-459).
In this SEB titration run, Abl used at 5 ng/well, was incubated 30 min with substrate-biotin (1 µM), and a non-limiting ATP concentration (100 µM) in a final assay volume of 20 µl. SEB concentrations ranging from 0 nM to 50 nM were tested. As shown here, SEB enhances S/B quite significantly.
In this second experiment, Abl was used at concentrations ranging from 0.10 to 10 ng/ml under similar substrate and ATP conditions as previously described, and with either 2.5 or 5 nM SEB. The subsequent S/B improvement at increasing SEB levels makes the use of lower tyrosine kinase concentration possible.
EnzymeCompoundTyrosine KinEASE assay
With SEB 50 nMWithout SEB
AblStaurosporine0.108-
Gleevec0.043-
CSKStaurosporine0.4930.417
Nexavar4.2206.175
AGFRStaurosporine1.0690.861
Iressa0.0010.002
JAK2Staurosporine0.000300.00035
Sutent0.8000.778
JAK3Staurosporine0.0010.001
Sutent7.6394.597
PTK5Staurosporine0.0390.036
Nexavar>0.1>0.1
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