Kinase toolbox reagents

A panel of sensitive and specific toolbox reagents for Tyr and Ser/Thr kinase screening.

Cisbio offers a line of ready to use toolbox reagents for the construction of Tyrosine and Serine/Threonine kinase activity assays. Our partnership with Cell Signaling Technology, a producer of high quality phospho-specific antibodies, means we can 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, HTRF Services also offers our expertise in custom labeling and kinase assay development. A real technical booster when you don't have the time to develop a kinase assay on your own - or for a highly specialized kinase assays.

Assay Principle

Cisbio offers an extensive list of both anti-phospho serine/threonine antibodies and 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 gives researchers the flexibility to create highly specific custom kinase assays. The assay below 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.

Product Performances

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, which means reducing reagent usage and enzyme consumption without sacrificing assay performance. Figure 1 shows the results in terms of S/B and Z' values for 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 learn more about the use of natural substrates, contact Cisbio Services.

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 performances 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).

Apparent ATP Km values determined by different COT assay formats

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

Membrane friendly

HTRF toolbox reagents are highly robust and can be used in assays with complex samples such as whole cells. Figure 3 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: 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. Poster by B. Bader et al. SBS 9th Annual Conference. September 2003, Portland (USA).

In addition to fresh and frozen cells, the HTRF Kinase toolbox can be used with membrane preparations. Figure 4 depicts an assay scheme for measuring autophosphorylation of a receptor tyrosine kinase (RTK), using membrane preparations as a source of active kinase.

Figure 4: Autophosphorylation of an RTK expressed as a tagged fusion protein is detected using a Eu3+ labeled anti-phospho tyrosine antibody and XL665 labeled anti-tag antibody.

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 Figure 5, HTRF tolerates the extremely high ATP concentrations (up to mM range) needed for mechnistic studies, e.g. the confirmation of ATP competitive inhibitors.

Figure 5: 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. Poster by B. Bader et al. SBS 9th Annual Conference. September 2003, Portland (USA).

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
PolyGT-Biotin - 5,000 tests61GT0BLA
PolyGT-Biotin - 100,000 tests61GT0BLD
PAb Anti-phospho PKA substrate-Eu cryptate - 500 tests61P01KAE
PAb Anti-phospho PKA substrate-Eu cryptate - 100,000 tests61P01KAY
PAb Anti-phospho PKA Substrate-Eu cryptate - 10,000 tests61P01KAZ
PAb Anti-phospho AKT substrate-Eu cryptate - 500 tests61P02KAE
PAb Anti-phospho AKT substrate-Eu cryptate - 100,000 tests61P02KAY
PAb Anti-phospho AKT Substrate-Eu cryptate - 10,000 tests61P02KAZ
MAb Anti-phospho PKC substrate-Eu cryptate - 500 tests61P03KAE
MAb Anti-phospho PKC substrate-Eu cryptate -100,000 tests61P03KAY
MAb Anti-phospho PKC substrate-Eu cryptate - 10,000 tests61P03KAZ
MAb Anti-phospho P38-Eu cryptate - 500 tests61P04KAE
MAb Anti-phospho P38-Eu cryptate - 100,000 tests61P04KAY
MAb Anti-phospho P38-Eu cryptate - 10,000 tests61P04KAZ
MAb Anti-phospho I kappa B alpha-Eu cryptate - 500 tests61P06KAE
MAb Anti-phospho I kappa B alpha-Eu cryptate - 100,000 tests61P06KAY
MAb Anti-phospho I kappa B alpha-Eu cryptate - 10,000 test61P06KAZ
MAb Anti-phospho histone H3-Eu cryptate - 500 tests61P07KAE
MAb Anti-phospho histone H3-Eu cryptate - 100,000 tests61P07KAY
MAb Anti-phospho histone H3-Eu cryptate - 10,000 tests61P07KAZ
MAb Anti-phospho p53-Eu cryptate - 500 tests61P08KAE
MAb Anti-phospho p53-Eu cryptate - 100,000 tests61P08KAY
MAb Anti-phospho p53-Eu cryptate - 10,000 tests61P08KAZ
MAb Anti-phosphoSAP/JNK-Eu cryptate - 500 tests61P09KAE
MAb Anti-phosphoSAP/JNK-Eu cryptate - 100,000 tests61P09KAY
MAb Anti-phosphoSAP/JNK-Eu cryptate - 10,000 tests61P09KAZ
MAb Anti-phosphop44/42-Eu cryptate - 500 tests61P10KAE
MAb Anti-phosphop44/42-Eu cryptate - 100,000 tests61P10KAY
MAb Anti-phosphop44/42-Eu cryptate - 10,000 tests61P10KAZ
MAb Anti-phospho ELK-1-Eu cryptate - 500 tests61P11KAE
MAb Anti-phospho ELK-1-Eu cryptate - 100,000 tests61P11KAY
MAb Anti-phospho ELK-1-Eu cryptate - 10,000 tests61P11KAZ
PAb Anti-phospho ATF2-Eu cryptate - 500 tests61P12KAE
PAb Anti-phospho ATF2-Eu cryptate - 100,000 tests61P12KAY
PAb Anti-phospho ATF2-Eu cryptate - 10,000 tests61P12KAZ
MAb Anti-phospho CREB-Eu cryptate - 500 tests61P13KAE
MAb Anti-phospho CREB-Eu cryptate - 100,000 tests61P13KAY
MAb Anti-phospho CREB-Eu cryptate - 10,000 tests61P13KAZ
PAb Anti-phospho C-JUN-Eu cryptate - 500 tests61P14KAE
PAb Anti-phospho C-JUN-Eu cryptate - 100,000 tests61P14KAY
PAb Anti-phospho C-JUN-Eu cryptate - 10,000 tests61P14KAZ
MAb Anti-phospho AKT-Eu cryptate - 500 tests61P15KAE
MAb Anti-phospho AKT-Eu cryptate - 100,000 tests61P15KAY
MAb Anti-phospho AKT-Eu cryptate - 10,000 tests61P15KAZ
PAb Anti-phospho MEK1/2-Eu cryptate - 500 tests61P17KAE
PAb Anti-phospho MEK1/2-Eu cryptate - 100,000 tests61P17KAY
PAb Anti-phospho MEK1/2-Eu cryptate - 10,000 tests61P17KAZ
PAb Antiphosphothreonine-Eu cryptate - 500 tests61PTRKAE
PAb Antiphosphothreonine-Eu cryptate - 100,000 tests61PTRKAY
PAb Antiphosphothreonine-Eu cryptate - 10,000 tests61PTRKAZ
MAb PT66-Eu cryptate - 5,000 tests61T66KLA
MAb PT66-Eu cryptate - 20,000 tests61T66KLB
MAb PY20-Eu cryptate - 5,000 tests61Y20KLA
MAb PY20-Eu cryptate - 20,000 tests61Y20KLB

Companion products

DescriptionCat. noProduct insertMSDS
Streptavidin-d2 - 5,000 tests610SADLA
Streptavidin-d2 - 20,000 tests610SADLB
Streptavidin-d2 - 1,000 tests610SADLF
Streptavidin-XL665 - 5,000 tests610SAXLA
Streptavidin-XL665 - 20,000 tests610SAXLB
Streptavidin-XL665 - 1,000 tests610SAXLF
Streptavidin-XLent! - 5,000 tests611SAXLA
Streptavidin-XLent! - 20,000 tests611SAXLB

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