Kinase Toolbox Reagents
Cisbio offers a line of ready to use toolbox reagents for construction of Tyrosine and Serine/Threonine kinase activity assays. Our partnership with Cell Signaling Technology, a producer of high quality phospho-specific antibodies, allows us to offer our customers a wide range of HTRF europium-cryptate labeled compatible antibodies. Our toolbox reagents also include XL665 conjugates and various kinase substrates.
If you need specific tools for your kinase of interest, Cisbio services also offers our expertise in custom labeling and kinase assay development. A real techinical booster when you don't have the time to develop a kinase assay on your own - or for a highly specialized kinase assays (learn more)!
- Able to use peptide or protein kinase substrates
- Flexibility in substrate labeling (biotin, tag, DNP, FLAG)
- Extensive line of pre-labeled Tyr & Ser/Thr antibodies
- No limitation in ATP concentrations
- Easily miniaturized to <10µL
Specific kinase assay developments using kinase toolbox
Ser/Thr kinases: AMPK alpha, Akt1/PKB alpha, Akt1/PKB alpha, Aurora B, Cam kinase II, CDK1/cyclin B, CDK2/cyclin E, CDK2, CDK4/cyclin B, CDK5, CHK1, CK2 alpha, DAPK1, ERK2, GSK3beta, HIPK2, IGF1-r, IKK beta, IRAK4, JNK1, JNK2, MAPKAPK2, MAPKAPK3, MAPKAPK5, MARK1, MEK1, MKK6, MSK1, NEK2, PAK2, PDK1, PIM1, PKA, PKC alpha, PKC beta1, PKCbeta2, PKC delta, PKC gamma, PKC théta, PKD2, PKG béta1, P38 alpha, P38 beta 2, P55 Fyn, P70S6K, RAF 1, ROCK II, RSK 2, SGK1
Tyr kinases: CSK, c-Met, EGFR, EPHB4, FAK, FES, FGR1, JAK3, LynA, PDGFR beta, Src, SYK, Tie2, TRK A, VEGFR1 (=FLT1), VEGFR2 (=KDR)
Assay Principle:
Cisbio offers more than 17 anti-phospho serine/threonine antibodies and 5 anti-phospho tyrosine antibodies labeled with Eu 3+- Cryptate, as well as XL-665 conjugates (Streptavidin, anti-6His, Anti-GST, etc.) and biotinylated Tyr kinase substrates. This comprehensive line of reagents provides researchers the flexibility to create highly specific custom kinase assays. This assay scheme depicts a typical kinase assay using a combination of a Eu 3+-Cryptate labeled anti-phosphoresidue antibody, SA-XL665 and a biotinylated substrate. The signal is proportional to the concentration of phospho-residues. Alternatively, other tags such as 6HIS, GST, c-myc, DNP, or FLAG may be used to label the kinase substrate.
Assay Performance:
HTRF Kinase toolbox reagents produce quality data with high signal:background (S/B) ratios, high Z' factors and low CVs. Unlike other technologies, HTRF allows true assay miniaturization, giving reduction of reagent usage and enzyme consumption without sacrificing assay performance. Figure 1 shows the results in terms of S/B and Z' values of a side-by-side comparison of a tyrosine kinase assay using HTRF technology or SPA.
Figure 1: HTRF(A) vs. SPA(B) assay performance. HTRF detection used Eu3+-cryptate labeled PT66 (0.13µg/ml) and SA XL665 (1.6µg/ml). SPA detection used SA-coated SPA beads (70 g beads/well) from Amersham Bioscience and 33P-ATP (6nCi/well). Biotinylated peptide, tyrosine kinase and ATP were used for both methods. For detailed protocols please refer to: Bregsdorf C, Bõmer U, SBS 9th Annual Conference. September 2003, Portland (USA).
Multiple Assay Format:
Working with natural substrates:
The HTRF Kinase toolbox reagents enable a high level of assay format flexibility and can be used with any tagged peptide or protien substrates, as shown in Figure 2. Other HTRF assays have been developed using substrates such as AFT2 (GST or Biotin tagged), CREB, PKC. To hear more about the use of natural substrates, contact Cisbio services.
| Assay Format | Apparent ATP Km(µM) |
| COT cascade | ND |
| COT/MBP protein direct33P | 384 ± 55 |
| COT/MEK protein direct HTRF | 279 ± 40 |
| COT/MEK peptide direct HTRF | 268 ± 21 |
Figure 2: Development of COT assay using direct and cascade format for HTS and mechanistic studies. Cascade can be useful when the direct anti-phospho substrate antibodies are not available. The performance of each format were compared to P33 filter-binding assay, to confirm enzyme kinetics and inhibitor profile. A study published by J. Jia (Anal. Biochem. 2006:305:268-276).
Membrane friendly
The HTRF Kinase toolbox reagents can be used in combination with fresh or frozen cells in addition to membrane preparations. Figure 3 depicts an assay scheme for measuring autophosphorylation of a receptor tyrosine kinase (RTK), using membrane preparations as a source of active kinase.
HTRF toolbox reagents are highly robust and can be used in assays with complex samples such as whole cells. Figure 4 summarizes the results of a study evaluating the compatibility of different cell lysis buffers with an RTK activation assay. Z' factors > 0.8 were seen for all 3 buffers.
Figure 3: Autophosphorylation of a RTK expressed as a tagged fusion protein is detected using a Eu3+ labeled anti-phospho tyrosine antibody and XL665 labeled anti-tag antibody.
Figure 4: Cells were stimulated with (black) or without (grey) a known RTK agonist. Cells were lysed using one of three buffers and phosphorylation levels assessed using a Eu3+ labeled anti-phospho tyrosine antibody and an anti-Myc antibody labeled with XL665. A poster from B. Bader and all. SBS 9th Annual Conference. September 2003, Portland (USA)
Detection at Physiological ATP Concentration:
ATP concentration is another important factor for in vitro kinase assay design, as the sensitivity of ATP competitive inhibitors varies wtih ATP concentration. As shown in fig. 5, HTRF tolerates the extremely high ATP concentrations (up to mM range) needed for mechnistic studies, e.g. the confirmation of ATP competitive inhibitors.
ATP concentration: A: 5000 µM, B: 500 µM, C: 50 µM, D: 5 µM, E: 0.5 µM (ATP Km)
Fig. 5: ATP competitor identification. Inhibitor dose reponse curves were generated using 15pg/µl Tyr enzyme, 0.3 µM of Tyr substrate and various ATP concentrations in 15 µL. Following incubation period, phospho-substrate is detected by a mixture of SAXL-665 and Eu-Cryptate labeled PT66. The sensitivity of ATP competitive inhibitors decreases with increasing ATP concentrations, meaning an increase of IC50 values.
Partnership with Cell Signaling Technology:
Cisbio and Cell Signaling Technology have come together to offer high quality anti-phospho serine/threonine antibodies (target and motif specific antibodies) labeled with Eu3+Cryptate, the HTRF donor fluorophore. Extensive evaluation of each Eu3+Cryptate labeled antibody's performances were conducted using the appropriate phosphorylated peptide (table 3). As an example the validation of the Phospho-p38 MAPK (Thr180/Tyr182) 28B10 monoclonal antibody is shown in Figure 6. Note the low CVs observed during the validation study.
www.cellsignal.com
PI 3-kinase Screening:
Millipore (Upstate) and Cisbio have united their technologies to create a high quality PI3 kinase screening service. The PIProfiler™ testing service includes the HTRF® based PI 3-K assay. This is a competitive assay which uses biotinylated PIP3 substrate and GST-tagged GRP1 PH domain. Assay detection uses streptavidin-XL665 and an anti-GST Eu3+-Cryptate labeled antibody. More information is available at www.millipore.com.
Ordering Info:
Ser/Thr Kinase Reagents:
| Reagent | Vendor # | Phospho-peptide control (motif) | Size (tests) | Qty/vial (µg) | Part # | Product Insert | MSDS |
| PAb Anti-phospho-(Ser/Thr) PKA-substrate -KA | CST # 9621 | CREB (Ser133) (RxxT*xx / RRxS*xx) |
500 10,000 100,000 |
0.5 10 100 |
61P01KAE 61PO1KAZ* 61PO1KAY* |
Request MSDS | |
| PAb Anti-phospho-(Ser/Thr) Akt-substrate-K | CST#9611 | PKA (Thr197) (R/KxR/KxxS*/T*) |
500 10,000 100,000 |
1.25 25 250 |
61P02KAE 61P02KAZ* 61P02KAY* |
Request MSDS | |
| PAb Anti-phospho-(Ser) PKC-substrate-K | CST#2261 | PLK (Ser137) (R/KxS-Hyd-R/K) |
500 10,000 100,000 |
0.75 15 150 |
61P03KAE 61P03KAY* 61P03KAZ* |
Request MSDS | |
| MAb Anti-phospho-p38 MAPK (Thr180/Tyr182) -K | CST#9216 | p38 MAPK (Thr180/Tyr182) | 500 10,000 100,000 |
1.25 25 250 |
61P04KAE 61P04KAZ* 61P04KAY* |
Request MSDS | |
| MAb Anti-phospho-IkappaB-alpha (Ser32/36)-K | CST#92461 | IkappaB-alpha (Ser32) | 500 10,000 100,000 |
0.25 5 50 |
61P06KAE 61P06KAZ* 61P06KAY* |
Request MSDS | |
| MAb Anti-phospho-Histone H3 (Ser10)-K | CST#9706 | Histone H3 (Ser10) | 500 10,000 100,000 |
1.25 25 250 |
61P07KAE 61P07KAZ* 61P07KAY* |
Request MSDS | |
| MAb Anti-phospho-p53 (Ser15) -K | CST#9286 | p53 (Ser15) | 500 10,000 100,000 |
1.5 30 300 |
61P08KAE 61P08KAZ* 61P08KAY* |
Request MSDS | |
| MAnti-phospho-SAPK/JNK (Thr183/Tyr185)-K | CST#9255 | SAPK/JNK (Thr183/Tyr185) | 500 10,000 100,000 |
0.5 10 100 |
61P09KAE 61P09KAZ* 61P09KAY* |
Request MSDS | |
| MAb Anti-phospho p44/42 MAPK (Thr202/Tyr204)-K | CST#9106 | p44/42 MAPK (Thr202/Tyr204) | 500 10,000 100,000 |
1 20 200 |
61P10KAE 61P10KAZ* 61P10KAY* |
Request MSDS | |
| MAb Anti-phospho-Elk-1 (Ser383)-K | CST#9186 | EIk-1 (Ser383) | 500 10,000 100,000 |
2.5 25 250 |
61P11KAE 61P11KAZ* 61P12KAY* |
Request MSDS | |
| PAb Anti-phospho-ATF-2 (Thr71)-K | CST#9221 | ATF2 fusion protein [CST#9224] |
500 10,000 100,000 |
2.5 50 |
61P12KAE 61P12KAZ* 61P12KAY* |
Request MSDS | |
| MAb Anti-phospho-CREB (Ser133)-K | CST#9196 | CREB (Ser133) | 500 10,000 100,000 |
0.25 5 50 |
61P13KAE 61P13KAZ* 61P13KAY* |
Request MSDS | |
| PAb Anti-phospho-c-Jun (Ser73)-K | CST#9164 | c-Jun (Ser73) | 500 10,000 100,000 |
1.25 25 250 |
61P14KAE 61P14KAZ* 61P14KAY* |
Request MSDS | |
| MAb Anti-phospho-Akt (ser473)-K | CST#4051 | Akt(Ser473) | 500 10,000 100,000 |
1.25 25 250 |
61P15KAE 61P15KAZ* 61P15KAY* |
Request MSDS | |
| PAb Anti-phosphothreonine-K | CST#9381 | PKA (Thr197) | 500 10,000 100,000 |
0.25 5 50 |
61PTRKAE 61PTRKAZ* 61P15KAY* |
Request MSDS | |
| PAb Anti-phospho MEK1/2 (Ser 217/221)-K | CST#9121 | MEK1/2 (Ser217/221) | 500 10,000 100,000 |
1.5 30 300 |
61P17KAE 61P17KAZ* 61P17KAY* |
Request MSDS | |
| * on request | |||||||
Tyrosine Kinase Reagents:
| Reagent | Vendor # | Phospho-peptide control (motif) | Size (tests) | Qty/vial (µg) | Part # | Product Insert | MSDS |
| MAb PT66-K | Poly-(GAT) or poly-(GT)-biotin | 5,000 20,000 |
7.5 30 |
61T66KLA 61T66KLB |
Request MSDS | ||
| MAb PY20-K | Poly-(GAT) or poly-(GT)-biotin | 5,000 20,000 |
10 40 |
61Y20KLA 61Y20KLB |
Request MSDS | ||
| * on request | |||||||
Tyrosine Kinase Substrates:
| Reagent | Size* (tests) | Part # | Qty/vial (µg)(tests) | Product Insert | MSDS |
| Poly-(GT)-biotin | 5,000 100,000 |
61GT0BLA 61GT0BLD |
250 5,000 |
Request MSDS | |
| Poly-(GAT)-biotin | 5,000 20,000 |
61GATBLA 61GATBLB |
250 1,000 |
Request MSDS |
Companion reagents:
| Sa-XLent! Sa-XL665 |
Affinity System Reagents |
| Anti-6HIS-XL665 Anti-GST-XL665 |
Anti-tag Reagents |
Kinase buffers:
| Reagent | Size | Part # |
| Enzymatic Buffer/kinase 5x | 50mL | 62EZBFDD |
| HTRF Detection buffer | 200mL | 62SDBRDF |
Depending on our stocks, some antibodies can be supplied immediately as bulk batches. Alternatively, should an antibody not be found in our list or should its stock be insufficient for your needs, it can be supplied as a cryptate custom labeling. Please contact us to find out about batch availability.
For more information or To Place an Order:
Europe and other countries
+33(0)466 796 705
