BindingDB logo
myBDB logout

Assay Method Information

Assay Name:  PI3-Kinase HTRF Assay
Description:  For the assay 50 nL of a 80-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 3 μl of a solution of PI3Kβ and phosphatidylinositol-4,5-bisphosphate (PIP2, 13.8 μM=>final conc. in 4 μl reaction volume=10 μM) in 1× reaction buffer (exact composition not disclosed by the vendor) were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. The amount of PI3Kβ was chosen to have the enzyme reaction in the linear range and depended on the activity of the individual lot, typical concentrations in assay were in the range of 120 ng/mL. Then the kinase reaction was started by the addition of 1 μL of a solution of adenosine triphosphate (ATP, 40 μM=>final conc. in the 4 μl assay volume is 10 μM) in reaction buffer and the resulting mixture was incubated for a reaction time of 20 min at 22° C. The reaction was stopped by the addition of 1 μL of an stop solution (containing the biotinylated PIP3 used as a tracer). Then 1 μL detection mix (containing a Europium-labeled anti-GST monoclonal antibody, a GST-tagged PH domain, and Streptavidin-Allophycocyanin) was added and resulting mixture was incubated for 3 h at 22° C. to allow the formation of complexes between the detection reagents and either the PIP3 generated in the kinase reaction, or the biotinylated PIP3 added with the stop solution. Subsequently the amount of energy transfer complex consisting of a Europium-labeled anti-GST monoclonal antibody, a GST-tagged PH domain, biotinylated PIP3 and Streptavidin-Allophycocyanin (APC) was evaluated by measurement of the resonance energy transfer from the Europium-labeled anti-GST monoclonal antibody to the Streptavidin-Allophycocyanin. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured using a TR-FRET reader, e.g., a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm were taken as the measure for the amount of biotinylated PIP3 bound to the GST-tagged PH domain, which is negatively correlated with the amount of PIP3 generated. The data were normalized (enzyme reaction without inhibitor=0% inhibition, all other assay components in the absence of enzyme=100% inhibition). Normally, test compounds were tested on the same microtiter plate at 10 different concentrations in the range of 25 μM to 1.3 nM (25 μM, 8.3 μM, 2.8 μM, 0.93 μM, 0.31 μM, 103 nM, 34 nM, 11 nM, 3.8 nM and 1.3 nM, a dilution series prepared before the assay at the level of the 80-fold conc. stock solutions by serial 1:3 dilutions) in duplicate values for each concentration and IC50 values were calculated by a 4 parameter fit using in-house software.
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail
   
    

Home

|

Search

|

Deposit

|

SiteMap

|

About us

|

Email us

|

Info

 
Last update November 1, 2007
©2000 BindingDB. All rights reserved.