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HTRF Human and Mouse Phospho-YES Tyr426 Detection Kit, 10,000 assay points

YES Phospho-Tyr426 Product image
HTRF Human and Mouse Phospho-YES Tyr426 Detection Kit, 10,000 assay points
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image

This HTRF kit allows for the cell-based quantitative detection of YES when phosphorylated at Tyr426.

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL
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This HTRF kit allows for the cell-based quantitative detection of YES when phosphorylated at Tyr426.

View product information
Product Variants
Unit Size: 500 assay points
Part #:
64YESY4PEG
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
HTRF Human and Mouse Phospho-YES Tyr426 Detection Kit, 10,000 assay points
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image
HTRF Human and Mouse Phospho-YES Tyr426 Detection Kit, 10,000 assay points
YES Phospho-Tyr426 Product image
YES Phospho-Tyr426 Product image

Product information

Overview

YES, also known as YES1 which belongs to the Src family of kinases, is a proto-oncogene and a non-receptor tyrosine kinase located at the plasma membrane. Once activated, YES phosphorylates various downstream effectors including LATS1/2, YAP, FAK, Caveolin-1, and p130CAS, as well as Crk. The kinase contributes to a wide range of cellular processes, including cell proliferation, survival, cytoskeleton rearrangements, migration, and adhesion. Dysregulation of YES can contribute to pathological conditions, especially in the context of cancer.

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 4
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
YES
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
TR-FRET
Therapeutic Area
Inflammation
Oncology
Unit Size
10,000 assay points

How it works

Phospho-YES (Tyr426) assay principle

The phospho-YES (Tyr426) assay measures YES when phosphorylated at Tyr26. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation leads to an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

generic-R-D-attributes-assay-principle-phospho-with-total.png
Phospho-YES (Tyr426) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-YES (Tyr426) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

generic-R-D-attributes-assay-protocol-how-it-works-2-plates.png

 

Phospho-YES (Tyr426) one-plate assay protocol

Detection of phosphorylated-YES(Tyr426) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality.

generic-R-D-attributes-assay-protocol-how-it-works-1-plate.png

Assay validation

Inhibition of phospho-YES (Ty426) in HEK293 cells

HEK293 cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 1.5 h with increasing concentrations of Dasatinib, and 30 µM of Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-YES (Tyr426) or Total-YES detection reagents were added. The HTRF signal was recorded after 4h.

As expected, the Src kinase inhibitor Dasatinib induced a dose-dependent decrease in YES phosphorylation, without impacting the expression level of the protein, while no toxicity was detected (ATPlite Luminescence Assay System, #6016943).

assay-validation-inhibitor-dasatinib-YES-total-64YESTPEG-64YESTPEH-64YESTPEY.jpg
assay-validation-degrader-dose-response-YES-total-64YESTPEG-64YESTPEH-64YESTPEY.jpg

HEK293 cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 1.5h with increasing concentrations of CH6953755, and 30 µM of Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-YES (Tyr426) or Total-YES detection reagents were added. The HTRF signal was recorded after 4h.

As expected, the Src kinase inhibitor CH6953755 induced a dose-dependent decrease in YES phosphorylation, without significantly impacting the expression level of the protein, while no toxicity was detected (ATPlite Luminescence Assay System, #6016943).

 

Assessment of phospho-YES (Tyr426) level in various cell lines

HEK293, HeLa, HAP1, and A431 human cells and NIH3T3 mouse cells were seeded at 100,000 cells/well in a 96-well microplate. After 24h of incubation, the cells were treated for 30 min with 30 µM of Pervanadate before being lysed for 30 minutes with supplemented lysis buffer #4, following the protocol for adherent or suspended cells, at RT under gentle shaking.
16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho-YES (Tyr426) detection reagents. The HTRF signal was recorded after a 4h incubation.
The HTRF Phospho-YES (Tyr426) assay detected YES phosphorylation at residue Y426 in various cellular models with different phosphorylation levels.

assay-validation-versatility-YES-phospho-Y426-64YESY4PEG-64YESY4PEH-64YESY4PEY.jpg
HTRF YES Phospho-Y426 assay compared to Western Blot

HEK293 cells were grown in a T175 flask in complete culture medium at 37°C, 5% CO2 until 80% confluence. 30µM of Pervanadate were added to the cells for 30 minutes before being lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 13 µL of each dilution were transferred for both detections. For HTRF detection, 13µL were transferred into a white 384-well microplate before the addition of 3 µL of supplemented lysis buffer #4 (1X). 4 µL of HTRF Total-YES detection reagents were then added. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Phospho-YES (Tyr426) assay, 1,950 cells/well were enough to detect a significant signal, while 7,800 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Phospho-YES (Tyr426) assay was 8 times more sensitive than the Western Blot technique.

assay-validation-WB-YES-phospho-Y426-64YESY4PEG-64YESY4PEH-64YESY4PEY.jpg

Simplified pathway

YES signaling pathway

YES, which belongs to the Src family of kinases, is a proto-oncogene and a non-receptor tyrosine kinase located at the plasma membrane. Once activated, YES phosphorylates various downstream effectors including LATS1/2, YAP, FAK, Caveolin-1, and p130CAS, as well as Crk. The kinase contributes to a wide range of cellular processes, including cell proliferation, survival, cytoskeleton rearrangements, migration, and adhesion. Dysregulation of YES can contribute to pathological conditions, especially in the context of cancer.

Simplified-pathway-YES-phospho-Y426-64YESY4PEG-64YESY4PEH-64YESY4PEY.jpg

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Technical Note
Guidelines for optimizing tyrosine phosphorylation detection: Effective use of pervanadate in kinase small molecule inhibitors studies

This technical note explores how pervanadate enhances tyrosine phosphorylation detection, enabling clearer insights into kinase...

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