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HTRF Human Total YES Detection Kit, 10,000 assay points

Total YES Product image
HTRF Human Total YES Detection Kit, 10,000 assay points
Total YES Product image
Total YES Product image
Total YES Product image
Total YES Product image
Total YES Product image

This HTRF kit allows for the cell-based quantitative detection of Total YES.

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MARCH10
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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 Total YES.

Click to copy promo code to clipboard.
March lab savings - SAVE 10%. Use promo code below.
MARCH10
Terms and conditions apply.
View product information
Product Variants
Unit Size: 500 assay points
Part #:
64YESTPEG
List Price
USD 2,147.00
Your online price:
Unit Size: 10,000 assay points
Part #:
64YESTPEH
List Price
USD 12,490.00
Your online price:
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).
Total list price:
USD 12,490.00
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Total YES Product image
Total YES Product image
HTRF Human Total YES Detection Kit, 10,000 assay points
Total YES Product image
Total YES Product image
Total YES Product image
HTRF Human Total YES Detection Kit, 10,000 assay points
Total YES Product image
Total YES 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
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
YES
Target Class
Phosphoproteins
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Inflammation
Oncology
Unit Size
10,000 assay points

How it works

Total YES assay principle

The Total YES assay quantifies the expression level of YES in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total YES assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of YES in a cell extract, these conjugates bring the donor fluorophore into close proximity with the acceptor, generating a FRET signal. The signal intensity is directly proportional to the concentration of the protein present in the sample and provides a means of assessing the protein's expression under a no-wash assay format

generic R D attributes assay principle total with phospho

 

Total YES 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 HTRF Total YES 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

 

Total YES one-plate assay protocol

Detection of Total YES 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 2 plates

 

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 CH6953755,

described to be an SF inhibitor, 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).

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

HeLa 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 4 and 24 hours with increasing concentrations of DAS-5-oCRBN, a Src family PROTAC. For the shortest treatment, a co-treatment with epoxomicin at 1µM was done.

The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, cell lysates were transferred into a 384-well low volume white microplate. 4 µL of the HTRF Total-YES or HTRF Total-c-Src detection reagents were added. The HTRF signal was recorded after 4h.

As expected, the Src Family PROTAC, DAS-5-oCRBN, induced a dose-dependent decrease in YES and c-Src expression. The kinetic of degradation was similar for both proteins. The presence of epoxomicin stopped the degradation, which demonstrated the proteasome involvement in the DAS-5-oCRBN induced process. Its effect was partial and increased over time. A slight decrease in cell viability was observed with the 24 hour treatment and on the highest PROTAC concentrations. After 4 hours of treatment, 25% of total YES and 53% of Src were degraded.

assay validation degrader dose response YES total 64YESTPEG 64YESTPEH 64YESTPEY 2
assay validation degrader dose response YES total 64YESTPEG 64YESTPEH 64YESTPEY 3

 

Validation of the selectivity of Total YES assay using siRNA Src Family

HAP1 cells were plated in a 96-well plate (100,000 cells/well) and cultured for 24h. The cells were then transfected with different siRNA, YES, Src, Fyn or Lyn, as well as with a negative control. Following a 24h incubation, 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 lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total YES detection antibodies were added. The HTRF signal was recorded after 4 hours.

Cell transfection with siRNA YES led to a significant decrease (70%) in the detection of the protein compared to the negative control (non targeting control). On the contrary, the transfection of other Src family members (Src, Lyn, and Fyn) did not induce any signal modulation, demonstrating that the HTRF Total YES assay is selective for YES and does not cross-react with other family members.

 

assay validation specificity YES total 64YESTPEG 64YESTPEH 64YESTPEY 2

 

Assessment of Total YES level in various cell lines

HEK293, HeLa, HAP1, and A431 human 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 Total-YES detection reagents. The HTRF signal was recorded after an 4h incubation.

The HTRF Total-YES assay detected the protein in human various cellular models with different expression levels.

 

assay validation versatility YES total 64YESTPEG 64YESTPEH 64YESTPEY 2

 

HTRF Total YES assay compared to Western Blot

Hela cells were grown in a T175 flask in complete culture medium at 37°C, 5% CO2 until 80% confluence. Cells were then 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 Total-YES assay, 1, 300 cells/well were enough to detect a significant signal, while 10,000 cells/well were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total-YES assay was 8 times more sensitive than the Western Blot technique.

 

assay validation WB YES total 64YESTPEG 64YESTPEH 64YESTPEY

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

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