Kinase Toolbox Reagents
Cisbio offers a line of 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 compatible, ready-to-use antibodies. Our toolbox reagents also include XL665 conjugates and various kinase substrates. Cisbio also offers custom labeling and assay development services for highly specialized kinase assays.
- 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
Assay Principle:
Cisbio offers 5 anti-phospho tyrosine antibodies and more than 17 anti-phospho serine/threonine antibodies labeled with Eu 3+-Cryptate as well as biotinylated Tyr kinase substrates and XL-665 conjugates (Streptavidin, anti-6His, Anti-GST,etc.). 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, biotinylated substrate and SA-XL665. 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 ratios, high Z' factors and low CVs. Unlike other technologies, HTRF allows true assay miniaturization, including reduction of reagent usage, without sacrificing assay performance. Figure 1 shows the results of a side-by-side comparison of a tyrosine kinase assay performed using HTRF technology or SPA. The final assay volume and plate type are indicated. The signal to background ratios were consistently higher using HTRF as well as the associated Z' factors. Of particular interest is that the absolute quantity of kinase and all HTRF detection reagents can be reduced upon miniaturization while SPA technology required maintenance of the quantity of radioactivity and SPA beads used per well.
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).
Membrane Compatibility:
The HTRF Kinase toolbox reagents enable a high level of assay flexibility and can be used in combination with fresh or frozen cells in addition to membrane preparations. Figure 2 depicts an assay scheme for measuring autophosphorylation of a receptor tyrosine kinase (RTK). The RTK is expressed as a fusion protein and membrane preparations as a source of active kinase. HTRF toolbox reagents such as europium labeled anti-phospho antibodies and XL665 labeled anti-tag antibodies then allow detection of kinase activity.
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 2: 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 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. A poster from B. Bader and all. SBS 9th Annual Conference. September 2003, Portland (USA)
Partnership with Cell Signaling Technology:
Cisbio and Cell Signaling Technology have come together to offer high quality anti-phospho serine/threonine
antibodies labeled with Eu3+Cryptate, the HTRF donor fluorophore. This line of labeled CST
antibodies ranges from target to phospho-motif specific antibodies (recognize a single phospho-residue) to motif specific
(recognize close epitopic sequence found in group of substrates). In addition, the phospho-threonine
antibodies bind phosphorylated threonine residues largely independently of the neighboring amino acid
sequence. The performance of each Eu3+Cryptate labeled antibody was validated using the appropriate
phosphorylated peptide. The validation of the Phospho-p38 MAPK (Thr180/Tyr182) 28B10 monoclonal antibody is
shown in Figure 4. Note the low CVs observed during the validation study.
www.cellsignal.com
Figure 4: The immunoreactivity of Eu3+-Cryptate labeled phospho-p38 MAPK (Thr180/Tyr182) 28B10 monoclonal antibody was validated by using various ratios of biotinylated -phosphopeptide to -peptide. The concentration of total peptide was kept constant (10 nM). Assay CVs are noted above the graph.
PI 3-kinase screening:
Upstate (part of Millipore) and Cisbio have joined 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.upstate.com.
Outsource your Kinase assay development to the experts:
Cisbio offers an assay development service as an attractive alternative to creating and optimizing custom
kinase assays within your own laboratory. Our dedicated teams work with you to develop an assay approach that
meets your specific needs.
More information »
Ordering Info:
Ser/Thr Kinase Reagents:
| Reagent | Vendor # | Phospho-peptide control (motif) | Size (tests) | Qty/vial (µg) | Part # | Product Insert |
| 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* |
|
| 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* |
|
| 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* |
|
| 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* |
|
| 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* |
|
| MAb Anti-phospho-Histone H3 (Ser10)-K | CST#9706 | Histone H3 (Ser10) | 500 10,000 100,000 |
1.25 25 250 |
61P07KAE 61P07KAZ* 61P07KAY* |
|
| MAb Anti-phospho-p53 (Ser15) -K | CST#9286 | p53 (Ser15) | 500 10,000 100,000 |
1.5 30 300 |
61P08KAE 61P08KAZ* 61P08KAY* |
|
| 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* |
|
| 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* |
|
| MAb Anti-phospho-Elk-1 (Ser383)-K | CST#9186 | EIk-1 (Ser383) | 500 10,000 100,000 |
2.5 25 250 |
61P11KAE 61P11KAZ* 61P12KAY* |
|
| 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* |
|
| MAb Anti-phospho-CREB (Ser133)-K | CST#9196 | CREB (Ser133) | 500 10,000 100,000 |
0.25 5 50 |
61P13KAE 61P13KAZ* 61P13KAY* |
|
| PAb Anti-phospho-c-Jun (Ser73)-K | CST#9164 | c-Jun (Ser73) | 500 10,000 100,000 |
1.25 25 250 |
61P14KAE 61P14KAZ* 61P14KAY* |
|
| MAb Anti-phospho-Akt (ser473)-K | CST#4051 | Akt(Ser473) | 500 10,000 100,000 |
1.25 25 250 |
61P15KAE 61P15KAZ* 61P15KAY* |
|
| MAb Anti-phosphothreonine-K | CST#9386 | PKA (Thr197) | 500 10,000 100,000 |
0.25 5 50 |
61P16KAE 61P16KAZ* 61P15KAY* |
|
| PAb Anti-phosphothreonine-K | CST#9381 | PKA (Thr197) | 500 10,000 100,000 |
0.25 5 50 |
61PTRKAE 61PTRKAZ* 61P15KAY* |
|
| 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* |
|
| * on request | ||||||
Tyrosine Kinase Reagents:
| Reagent | Vendor # | Phospho-peptide control (motif) | Size (tests) | Qty/vial (µg) | Part # | Product Insert |
| MAb PT100-K | CST#9411 | Poly-(GAT) or poly-(GT)-biotin | 500 10,000 |
0.5 10 |
61T00KAE 61T00KAZ* |
— |
| MAb PT102-K | CST#9416 | Poly-(GAT) or poly-(GT)-biotin | 500 10,000 |
0.25 5 |
61T02KAE 61T02KAZ* |
— |
| MAb PT66-K | Poly-(GAT) or poly-(GT)-biotin | 5,000 20,000 |
7.5 30 |
61T66KLA 61T66KLB |
||
| MAb PY20-K | Poly-(GAT) or poly-(GT)-biotin | 5,000 20,000 |
10 40 |
61Y20KLA 61Y20KLB |
||
| MAb 4G10-K | Upstate | Poly-(GAT) or poly-(GT)-biotin | 5,000 20,000 |
17.5 70 |
61G10KLA 61G10KLB |
|
| * on request | ||||||
Tyrosine Kinase Substrates:
| Reagent | Size* (tests) | Part # | Qty/vial (µg)(tests) | Product Insert |
| Poly-(GT)-biotin | 5,000 100,000 |
61GT0BLA 61GT0BLD |
250 5,000 |
|
| Poly-(GAT)-biotin | 5,000 20,000 |
61GATBLA 61GATBLB |
250 1,000 |
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:
U.S. and Canada
1-888-963-4567
Europe and other countries
+33(0)466 796 705
Japan
+81-3-5510-2932
