HTRF KinEASE-STK S3 kit HTRF®

The HTRF KinEASE assay series is a semi-universal biochemical platform to study kinase activity. The STK3 kit uses a specific substrate compatible with various Serine/thronine 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 STK S3 assay has been validated on 43 Serine/Threonine kinases. The assay validation section of this page lists them and provides technical tips for setup. The kit series also include a Discovery kit to help determine which of three STK kits is appropriate when working with a non-validated enzyme.

Benefits

COMPATIBLE WITH UP TO MM ATP KMS
SEMI-UNIVERSAL METHOD FOR STKS
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. All three STK peptidic substrates are made of the same sequence for antibody detection, next to a variable sequence recognized by enzymes. The targeted residue for phosphorylation lies in between as part of both sequences for enzymatic activity (unphosphorylated) and antibody detection (phosphorylated). It has been shown that 3 different variable sequences could enable the study of most Serine/Threonine kinases.

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 which use is recommended, and download the technical datasheet including experimental setting and validation data. Non listed STKs can be assessed with the Discovery kit to find out which HTRF KinEASE STK kit may be used.

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, 43 were found to be compatible with the S3 kit. The case of AKT1 Serine/Threonine kinase is illustrated below with the enzyme titration and time course (on the left), and the inhibition by staurosporine (on the right).