HTRF KinEASE-TK kit HTRF®

The HTRF KinEASE assay series is a semi-universal biochemical platform to study kinase activity. The TK kit uses a specific substrate compatible with tyrosine kinases - cf. validation data provided on this page.

Low enzyme consumption

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Overview

The HTRF KinEASE kit series provides a simple biochemical approach to study kinase activity, screen for inhibitors and characterize them. Three STK and one TK kits offer a semi-universal method for assessing phosphorylation on Serine/Threonine and Tyrosine residues, respectively. The HTRF KinEASE TK assay has been validated on 71 Tyrosine kinases, using a single generic substrate. The assay validation section of this page lists them and provides technical tips for setup.

Benefits

COMPATIBLE WITH UP TO MM ATP KMS
ONE SINGLE SUBSTRATE FOR ALL TKS
LOW ENZYME CONSUMPTION

Assay principle

Each HTRF KinEASE kit is based on the same assay format, and uses a universal biotinylated substrate, a phospho-specific Eu-cryptate-labeled monoclonal antibody, and SA-XL665. The TK generic substrate is made of a sequence of repetitive motifs recognized by most tyrosine kinases, and by the specific conjugated antibody once phosphorylated.

Assay protocol

As shown here, all HTRF KinEASE assays work the same way and involve two steps:

Step 1: Kinase reaction The kinase is incubated in the presence or absence of compounds and the appropriate substrate. The supplemented enzymatic buffer is added in the kinase buffer for tyr kinases only. ATP is added to start the reaction.

Step 2: Detection Eu-cryptate and XL665 conjugates are added. The detection buffer contains EDTA to stop the enzymatic reaction.

Click in the table below to view the list of enzymes, the kit whose use is recommended, and to download the technical datasheet including experimental setting and validation data.

ABL1 (TK)	ABL2 (TK)	AKT1 (STK S3)	AKT2 (STK S3)	AKT3 (STK S3)
ALK (TK)	AMPK a2/ß1/g1 (STK S1)	AMPK a2/ß2/g1 (STK S1)	Arg (TK)	ARK5 (STK S3)
ASK1 (STK S3)	AURKA (STK S2)	AURKB (STK S2)	Aurora A (STK S2)	Aurora B (STK S2)
Aurora C (STK S3)	AXL (TK)	BLK (TK)	BMX (TK)	BRK (TK)
BrSK1 (STK S3)	BrSK2 (STK S1)	BTK (TK)	c-Kit (TK)	CAMK1 (STK S1)
CaMK1 a (STK S1)	CaMK2 a (STK S1)	CaMK2 ß (STK S1)	CaMK2 d (STK S1)	CaMK2 g (STK S1)
CAMK2B (STK S1)	CAMK2D (STK S1)	CAMK2G (STK S1)	CaMK4 (STK S1)	CDC42 BPA (STK S3)
CDC42 BPB (STK S3)	CGK2 (STK S2)	CHEK1 (STK S1)	CHEK2 (STK S1)	CHK1 (STK S1)
CHK2 (STK S1)	CHUK (STK S3)	CLK3 (STK S3)	COT (STK S3)	CSF1R (TK)
CSK (TK)	DAPK1 (STK S1)	DAPK2 (STK S1)	DAPK3 (STK S1)	DCAMKL2 (STK S1)
DDR2 (TK)	DMPK (STK S3)	DRAK1 (STK S1)	DYRK2 (STK S3)	EEF-2K (STK S3)
EGFR (TK)	EPHA1 (TK)	EPHA2 (TK)	EPHA3 (TK)	EPHA4 (TK)
EPHA5 (TK)	EPHA7 (TK)	EPHA8 (TK)	EPHB1 (TK)	EPHB2 (TK)
EPHB3 (TK)	EPHB4 (TK)	ERBB4 (TK)	FAK (TK)	FER (TK)
FES (TK)	FGFR1 (TK)	FGFR2 (TK)	FGFR3 (TK)	FGFR4 (TK)
FGR (TK)	FLT1 (TK)	FLT3 (TK)	FLT4 (TK)	FMS (TK)
Fps (TK)	FRK (TK)	FYN (TK)	GPRK4 (STK S1)	GRK4 (STK S1)
GRK5 (STK S3)	GRK6 (STK S1)	GSG2 (STK S1)	GSK3 a (STK S3)	Haspin (STK S1)
HCK (TK)	HER4 (TK)	HIPK2 (STK S3)	HIPK3 (STK S3)	IGF1R (TK)
IKBKB (STK S3)	IKK a (STK S3)	IKK ß (STK S3)	INSR (TK)	INSRR (TK)
Insulin R (TK)	IRAK4 (STK S1)	IRR (TK)	ITK (TK)	JAK1 (TK)
JAK2 (TK)	JAK3 (TK)	KDR (TK)	KIT (TK)	LATS1 (STK S1)
LCK (TK)	LKB1 (STK S1)	LOK (STK S3)	Lyn (TK)	LYNa (TK)
MAP3K8 (STK S3)	MAPKAP-K1 a (STK S1)	MAPKAP-K1 ß (STK S1)	MAPKAP-K2 (STK S1)	MAPKAP-K3 (STK S1)
MAPKAP-K5 (STK S1)	MARK1 (STK S1)	MARK2 (STK S2)	MELK (STK S1)	MER (TK)
MERTK (TK)	MET (TK)	MINK1 (STK S3)	MKNK1 (STK S1)	MKNK2 (STK S1)
MLCK (STK S1)	MNK1 (STK S1)	MNK2 (STK S1)	MRCK a (STK S3)	MRCK ß (STK S3)
MSK1 (STK S3)	MSK2 (STK S3)	MSSK1 (STK S3)	MST1 (STK S3)	MST1R (TK)
MST2 (STK S3)	MSTIR (TK)	MUSK (TK)	MYLK (STK S1)	NEK11 (STK S2)
NEK2 (STK S3)	NEK3 (STK S3)	NEK6 (STK S3)	NEK7 (STK S3)	NLK (STK S3)
NTRK1 (TK)	NTRK2 (TK)	NTRK3 (TK)	p70S6K (STK S3)	PAK2 (STK S2)
PAK3 (STK S2)	PAK4 (STK S2)	PAK5 (STK S2)	PAK6 (STK S2)	PAK7 (STK S2)
PAR-1B a (STK S2)	PASK (STK S1)	PDGFR a (TK)	PDGFR ß (TK)	PDGFRA (TK)
PDGFRB (TK)	PHK g1 (STK S1)	PHK g2 (STK S1)	PIM1 (STK S3)	PIM2 (STK S3)
PKAC a (STK S2)	PKAC g (STK S3)	PKB a (STK S3)	PKB ß (STK S3)	PKB g (STK S3)
PKC a (STK S1)	PKC ß1 (STK S1)	PKC ß2 (STK S1)	PKC d (STK S1)	PKC epsilon (STK S1)
PKC µ (STK S1)	PKC theta (STK S1)	PKC eta (STK S1)	PKC gamma (STK S1)	PKC iota (STK S1)
PKC zeta (STK S1)	PKD2 (STK S1)	PKG1 a (STK S2)	PKG1 ß (STK S2)	PKG2 (STK S1)
PKN2 (STK S1)	PLK1 (STK S1)	PLK3 (STK S3)	PLK4 (STK S2)	PRAK (STK S1)
PRK2 (STK S1)	PRKA A2/B1/G1 (STK S1)	PRKACA (STK S2)	PRKACG (STK S3)	PRKCA (STK S1)
PRKCB1 (STK S1)	PRKCB2 (STK S1)	PRKCD (STK S1)	PRKCE (STK S1)	PRKCG (STK S1)
PRKCH (STK S1)	PRKCI (STK S1)	PRKCQ (STK S1)	PRKCZ (STK S1)	PRKD2 (STK S1)
PRKG1 (STK S2)	PRKG2 (STK S1)	PRKX (STK S2)	PTK2 (TK)	PTK2B (TK)
PTK5 (TK)	PTK6 (TK)	PYK2 (TK)	RET (TK)	ROCK1 (STK S2)
ROCK2 (STK S2)	ROK a (STK S2)	RON (TK)	ROS (TK)	ROS1 (TK)
RPS6KA1 (STK S1)	RPS6KA2 (STK S1)	RPS6KA3 (STK S1)	RPS6KA4 (STK S3)	RPS6KA5 (STK S3)
RPS6KA6 (STK S2)	RPS6KB1 (STK S3)	Rse (TK)	RSK1 (STK S1)	RSK2 (STK S1)
RSK3 (STK S1)	RSK4 (STK S2)	SAD1 (STK S3)	SGK1 (STK S3)	SGK2 (STK S3)
SGK3 (STK S3)	SGKL (STK S3)	SIK (STK S1)	smMLCK (STK S1)	Snk (STK S3)
SRC (TK)	STK22B (STK S1)	STK22D (STK S1)	STK23 (STK S3)	STK3 (STK S3)
STK4 (STK S3)	STK6 (STK S2)	SYK (TK)	TBK1 (STK S1)	TEK (TK)
TIE2 (TK)	TRKA (TK)	TRKB (TK)	TRKC (TK)	TSSK1 (STK S1)
TSSK2 (STK S1)	TYK2 (TK)	TYR03 (TK)	TYRO3 (TK)	VEGFR1 (TK)
VEGFR2 (TK)	VEGFR3 (TK)	WNK2 (STK S3)	WNK3 (STK S3)	YES1 (TK)
ZAP70 (TK)	ZIPK (STK S1)

Validated kinases

Over 190 Serine/Threonine and Tyrosine kinases have already been validated with the HTRF KinEASE kits. Out of those, 71 were tyrosine kinases that could be assessed with the TK kit. The case of PDGFRB Tyrosine kinase is illustrated below with the enzyme titration and time course (on the left), and the inhibition by staurosporine (on the right).