Kinase Toolbox Reagents

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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.

Features:
  • 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*
pdf
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*
pdf
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*
pdf
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*
pdf
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*
pdf
MAb Anti-phospho-Histone H3 (Ser10)-K CST#9706 Histone H3 (Ser10) 500
10,000
100,000
1.25
25
250
61P07KAE
61P07KAZ*
61P07KAY*
pdf
MAb Anti-phospho-p53 (Ser15) -K CST#9286 p53 (Ser15) 500
10,000
100,000
1.5
30
300
61P08KAE
61P08KAZ*
61P08KAY*
pdf
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*
pdf
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*
pdf
MAb Anti-phospho-Elk-1 (Ser383)-K CST#9186 EIk-1 (Ser383) 500
10,000
100,000
2.5
25
250
61P11KAE
61P11KAZ*
61P12KAY*
pdf
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*
pdf
MAb Anti-phospho-CREB (Ser133)-K CST#9196 CREB (Ser133) 500
10,000
100,000
0.25
5
50
61P13KAE
61P13KAZ*
61P13KAY*
pdf
PAb Anti-phospho-c-Jun (Ser73)-K CST#9164 c-Jun (Ser73) 500
10,000
100,000
1.25
25
250
61P14KAE
61P14KAZ*
61P14KAY*
pdf
MAb Anti-phospho-Akt (ser473)-K CST#4051 Akt(Ser473) 500
10,000
100,000
1.25
25
250
61P15KAE
61P15KAZ*
61P15KAY*
pdf
MAb Anti-phosphothreonine-K CST#9386 PKA (Thr197) 500
10,000
100,000
0.25
5
50
61P16KAE
61P16KAZ*
61P15KAY*
pdf
PAb Anti-phosphothreonine-K CST#9381 PKA (Thr197) 500
10,000
100,000
0.25
5
50
61PTRKAE
61PTRKAZ*
61P15KAY*
pdf
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*
pdf
* 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
pdf
MAb PY20-K   Poly-(GAT) or poly-(GT)-biotin 5,000
20,000
10
40
61Y20KLA
61Y20KLB
pdf
MAb 4G10-K Upstate Poly-(GAT) or poly-(GT)-biotin 5,000
20,000
17.5
70
61G10KLA
61G10KLB
pdf
* 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
pdf
Poly-(GAT)-biotin 5,000
20,000
61GATBLA
61GATBLB
250
1,000
pdf

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:
 

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