The HTRF KinEASE assay series is a semi-universal biochemical platform to study kinase activity. The Discovery kit provides a straightforward way to determine which of the STK kits must be used for a given serine/threonine kinase.
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  • Low enzyme consumption Low enzyme consumption
The HTRF KinEASE assay series is a semi-universal biochemical platform to study kinase activity. The Discovery kit provides a straightforward way to determine which of the STK kits must be used for a given serine/threonine kinase.
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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 (STK) and Tyrosine (TK) residues, respectively.

Over 188 kinases have been validated with this technique and the assay validation section of this page includes technical tips for each assay setup.

The Discovery kit is intended to help determine which of the three STK kits is appropriate when working with a non-validated Serine/Threonine kinase.

Benefits

  • EASY KINASE PRE-VALIDATION
  • 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 principle

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 Eu3+ cryptate and XL665 conjugates are added. The detection buffer contains EDTA to stop the enzymatic reaction.
HTRF KinEASE Assay principle

Validated kinases

Click in the table below to view the list of validated 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)
Over 190 Serine/Threonine and Tyrosine kinases have already been validated with the HTRF KinEASE kits, and their optimal substrate determined. Note that one given enzyme may be compatible with several substrates, as shown here with MST1 that work equally well with S3 and S2, but significantly less with S1.
MST1 kinase validation with HTRF KinEASE Discovery kit
HTRF KinEASE

Universal platform to address Ser/Thr & Tyr kinases - Application Notes

KinEASE, a universal expanded platform to adress Ser/Thr & Tyr kinases

Measuring kinase activity under any conditions - Application Notes

Exploration is easier with a trusted partner

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Cisbio KinEASE

Validated kinases and assay conditions - Flyers

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

The essential white paper to expand your knowledge on kinases

Learn about cutting edge kinase research in this free white paper - Guides

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