Product Pathways - Substrate Identification
Tyrosine Kinase Substrate Screening Kit #7450
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
| Kit Includes | Quantity |
|---|---|
| Phospho-Tyrosine Mouse mAb (P-Tyr-100) # 9411 | 60 microliters |
| Peptide Plates | 50 microliters |
| HTScan® Tyrosine Kinase Buffer (4X) # 9805 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| DTT (1000X, 1.25 M) | 80 microliters |
Description
The Tyrosine Kinase Substrate Screenining Kit provides 95 different nonphospho-peptides as kinase substrates. Also included are biotinylated phospho- and nonphospho-peptides of identical sequence to serve as positive and negative controls, respectively. Potential substrate phosphorylation is detected by phospho-tyrosine specific antibody #9411, provided in the kit.
Source / Purification
Detection antibody source: Phospho-Tyrosine mAb (P-Tyr-100) #9411 is derived from mice immunized with phospho-tyrosine-containing peptides coupled to KLH.
Quality Control
The quality of the peptides was evaluated by reverse-phase HPLC and mass spectrometry.
Specificity / Sensitivity
Phospho-Tyrosine mAb (P-Tyr-100) #9411 binds phospho-Tyr in a manner largely independent of the surrounding amino acid sequence, and does not cross-react with peptides containing phospho-Ser or phospho-Thr.DELFIA® assay yields 10 to over 100 fold signal-to-noise ratio of phospho- versus nonphospho-peptides .
Kinase Assay - Radiometric
Kinase of interest is incubated with biotinylated substrate peptides. Phosphorylated products are captured on streptavidin-coated plates. Cell Signaling Technology, Inc. antibody #9411 P-Tyr-100 serves as the primary detection reagent; DELFIA® Europium (Eu) anti-mouse chelates are used as secondary antibodies. Positive hits from Tyrosine Kinase Substrate Screening Kit #7450 can be used in any subsequent peptide based kinase assay.
Kinase Assay - DELFIA
Treatment of Tyrosine Kinase Substrate Screening Kit with Met kinase: Positive control is shown in yellow, and negative control is shown in blue. Several positive hits were identified using this approach (in red).
Kinase Assay - DELFIA
Treatment of Tyrosine Kinase Substrate Screening Kit with Src kinase: Positive control is shown in yellow, and negative control is shown in blue. Several positive hits were identified using this approach (in red).
Background
Numerous tyrosine kinases have become pharmaceutically important. With the cloning of hundreds of uncharacterized tyrosine kinases, drug discovery researchers are seeking to quickly find substrates for high throughput screening programs. To expedite the isolation of substrates for novel kinases, Cell Signaling Technology has developed kinase/substrate screening kits.With a total of 95 different peptides on the array, a very diverse group of substrates is represented. Signal-to-noise ratios have been optimized and range from 10 to over 100. The phosphotyrosine-specific antibody and peptide substrate can be used for subsequent high throughput peptide based kinase screens (e.g., FP, TRF, AlphaScreen).
Application References
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