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
The testing of compound-target engagement can be performed with our NanoBRET Target Engagement Intracellular Kinase Assay

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List of Cell-based Kinase Assays (57 total)

Target	HGNC name	Cell Line	Assay Format	Info sheet
AKT1	AKT1	Rat1	transfected	Data sheet
ALK	ALK	Karpas-299	endogenous	Data sheet
Aurora-B	AURKB	HT-29	endogenous	Data sheet
AXL	AXL	MEF	transfected	Data sheet
B-RAF-VE	BRAF (V600E)	Rat1	transfected	Data sheet
BCR-ABL	BCR-ABL1	K562	endogenous	Data sheet
EGF-R	EGFR	A431	endogenous	Data sheet
EGF-R (d746-750/C797S)	EGFR (d746-750/C797S)	Rat1	transfected	Data sheet
EGF-R (d746-750/T790M)	EGFR (d746-750/T790M)	Rat1	transfected	Data sheet
EGF-R (d746-750/T790M/C797S)	EGFR (d746-750/T790M/C797S)	Rat1	transfected	Data sheet
EGF-R (d747-749/A750P)	EGFR (d747-749/A750P)	Rat1	transfected	Data sheet
EGF-R (d752-759)	EGFR (d752-759)	Rat1	transfected	Data sheet
EGF-R (G719S)	EGFR (G719S)	Rat1	transfected	Data sheet
EGF-R (L858R)	EGFR (L858R)	Rat1	transfected	Data sheet
EGF-R (L861Q)	EGFR (L861Q)	Rat1	transfected	Data sheet
EGF-R (T790M)	EGFR (T790M)	Rat1	transfected	Data sheet
EGF-R (T790M/C797S/L858R)	EGFR (T790M/C797S/L858R)	Rat1	transfected	Data sheet
EGF-R (T790M/L858R)	EGFR (T790M/L858R)	Rat1	transfected	Data sheet
EGF-R (wild type control)	EGFR	Rat1	transfected	Data sheet
EPHB4	EPHB4	MEF	transfected	Data sheet
ERBB2	ERBB2	NIH3T3	transfected	Data sheet
ERBB4	ERBB4	T47D	endogenous	Data sheet
MEK1/2	MAP2K1/MAP2K2	PANC-1	endogenous	Data sheet
FAK	PTK2	MEF	transfected	Data sheet
FGF-R2	FGFR2	Kato III	endogenous	Data sheet
FLT3 (DY)	FLT3 (D835Y)	MEF	transfected	Data sheet
FLT3 (ITD)	FLT3 (ITD)	MEF	transfected	Data sheet
FLT3 (wild type control)	FLT3	MEF	transfected	Data sheet
FYN	FYN	Rat1	transfected	Data sheet
IGF1-R	IGF1R	MEF	transfected	Data sheet
KIT	KIT	M07e	endogenous	Data sheet
LRRK2	LRRK2	A549	endogenous	Data sheet
MET	MET	MKN45	endogenous	Data sheet
MET (D1228N)	MET (D1228N)	Rat1	transfected	Data sheet
MET (D1288H)	MET (D1288H)	Rat1	transfected	Data sheet
MET (F1200l)	MET (F1200I)	Rat1	transfected	Data sheet
MET (wild type control)	MET	Rat1	transfected	Data sheet
MET (Y1230A)	MET (Y1230A)	Rat1	transfected	Data sheet
MET (Y1230D)	MET (Y1230D)	Rat1	transfected	Data sheet
MET (Y1230H)	MET (Y1230H)	Rat1	transfected	Data sheet
PDGFR-beta	PDGFRB	NIH3T3	endogenous	Data sheet
PIM1	PIM1	HEK293T	transfected	Data sheet
PIM2	PIM2	HEK293T	transfected	Data sheet
PIM3	PIM3	HEK293T	transfected	Data sheet
ROCK	ROCK1 and ROCK2	A7r5	endogenous	Data sheet
RON	MST1R	T47D	endogenous	Data sheet
SRC	SRC	MEF	transfected	Data sheet
VEGF-R2	KDR	HUE	endogenous	Data sheet
VEGF-R3	FLT4	MEF	transfected	Data sheet
HPK1	MAP4K1	Jurkat	endogenous	Data sheet

Cell Phosphorylation Assay Details

Assay Procedure

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 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.

Please see our FAQ page to access our cell-based assay condition form (German facility) for further details on compound preparation and shipping.

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.

After heat inactivation of plasma and medium, dose-response curves could be generated in the cellular phosphorylation assay.

Left panel: The drug has a low plasma binding potential and it’s activity is only slightly affected by the presence of human plasma.
Right panel: The drug has a high plasma binding potential and the activity of the drug 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.

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

Cellular Phosphorylation Assay Services

Cellular Phosphorlyation Assay Principle

List of Cell-based Kinase Assays (57 total)

Cell Phosphorylation Assay Details

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