Cellular Phosphorylation Assay Services

The Cellular Phosphorylation Assay quantifies changes in the phosphorylation status of a substrate as a result of treatment with your inhibitor in intact cells. The assay was designed to address compound activity in a physiological environment on a physiological substrate and is one of two cell-based kinase assays we offer besides the NanoBRET Target Engagement Intracellular Kinase Assay

The assay is available with either an endogenously expressed target kinase or with transduced cells for overexpression of the kinase of interest. Cells are preincubated with the compound to allow thorough target binding. To perform substrate phosphorylation some kinases are constitutively active and some kinases need to be activated with incubation of a ligand. After stopping the reaction by cell lysis, the substrate phosphorylation is quantified via ELISA assay. 

  • Physiological kinase, substrate, and ATP concentrations
  • The assay can be performed with animal blood containing the drug for plasma-inhibitory study after in vivo administration
  • Deliverable: % inhibition of kinase activity and IC50 determination

Cellular Phosphorlyation Assay Principle

The Cellular Phosphorylation Assay is an activity assay directly measuring the capacity of a compound to inhibit kinase activity in the cellular setup.

The substrate is either an endogenous kinase-specific protein or the kinase performs autophosphorylation on itself.

principle of a kinase assay in intact cells

Assay Details

Assay Procedure
Assay procedure kinase assay in intact cells

Assay principle on the example of the VEGF-R2 assay.

Human endothelial cells express endogenous VEGF-R2. Compounds are added to the serum-free cell culture medium via nanodrop dispenser and the cells incubate with the test compound for 90 minutes to allow for target binding. After a 3-minute stimulation with ligand VEGF-A, cells are lysed and the substrate phosphorylation is quantified by ELISA with pan-phospho-tyrosine antibodies on captured VEGF-R2. The assay is performed with 8 compound concentrations in duplicate for IC50 value determination.

Mutant Panels
Assay principle dimerization of receptor tyrosine kinases in intact cells

Assay with transfected kinase constructs.

To provide a panel of kinase mutants we transfected fibroblasts to stably express the intracellular domain of receptor kinase mutants fused to an artificial transmembrane domain. Dimerization of the receptors causes constitutive auto-phosphorylation that can be interrupted by a specific kinase inhibitor and quantified via ELISA.

Mutant panels are available for EGF-R, MET and FLT3.

Screening details

Setups: IC50 value determination. 

Controls: DMSO only control serves as high control. Quality assurance is provided by calculation of Z' factors for low/high controls on each assay plate and by including a full IC50 curve of a kinase-specific reference inhibitor to monitor adequate dose/response relation in your assay run.

Custom assay development: Assays that are not part of the list can be established according to the client’s research needs. 

Turnaround time for standard studies is about 2 to 3 weeks.

Report: We provide a detailed report including materials, methods, raw data, and graphical presentation of IC50 dose-response curves.

Screening facility: The assay is performed in Freiburg, Germany. 

Compound requirements: Compound requirements in brief: 30 µl of the stock solution with 1000x of the highest assay concentration or the equivalent of solid material.

Plasma Protein Inhibitory Assay

The Plasma Inhibitory Assay (PIA) is used to determine the impact of the presence of plasma on the performance of kinase inhibitors. 

The PIA Assay can be performed with two setups:

In vitro: The cell-culture medium is supplemented with blood plasma during the performance of the Cellular Phosphorylation Assay. 

graphs displaying the effects of plasma binding to compounds and inhibit the activity of compounds

After heat inactivation of plasma and medium, dose-response curves could be generated in the cellular phosphorylation assay. The activity of Sunitinib as a molecule with low plasma binding is only slightly affected by the presence of human plasma. The activity of Sorafenib which has a high plasma binding is reduced more than 460fold in the presence of human plasma.

In vivo: The drug is administered to a mouse and its blood is used for quantification of the amount of functional inhibitor in the blood. 

plasma inhibitory effect measured in mice with kinase activity assay

A) The inhibitory effect on FLT3 autophosphorylation was assessed for plasmas from mice taken at four different time points after oral application of Sunitinib (80mg/kg). B) The dose-response curve for Sunitinib spiked in mouse plasma is shown as a control. C) The concentration of Sunitinib in mouse plasma after different time points.