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

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.

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.

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.

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.

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.

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.