Phospho-YAP (Ser127) & Total YAP Cellular Assay Kits

Phospho- and total YAP detection as a readout of the Hippo-YAP pathway

Based on our homogeneous and robust HTRF® technology, the phospho-YAP (Ser127) kit enables the detection of YAP (Yes-associated protein) phosphorylated at Serine 127 while the total YAP kit is designed for the detection of total YAP, phosphorylated and unphosphorylated. The buffers of both HTRF® phospho- and total YAP assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample. With its central mediator role in the Hippo Pathway, YAP is an important target for oncological disease studies.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total YAP assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-YAP antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with YAP, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-YAP can be quantitatively detected using the HTRF phospho-YAP (Ser127) and total YAP cellular assay kit reagents, and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

Hippo-YAP cell signaling

The Hippo/YAP pathway regulates organ size by playing a role in the balance between proliferation & apoptosis. In addition this pathway is involved in mechanical and cytoskeletal signal transduction. The Hippo/YAP pathway is activated by numerous stimuli, such as cell attachment, high cell density, mechanical tension, or in the absence of growth factors. The activation of this pathway first induces the activation of the kinase MTS, which in turn phosphorylates Lats kinases. YAP is then phosphorylated on Serine 127, leading to its cytoplasmic retention and eventually to its degradation by the proteasome machinery. Thus phosphorylated YAP represents the inactivated form of the protein.

When the Hippo/YAP pathway is inactivated, YAP/TAZ accumulates in the nucleus and induces the transcription of genes involved in cell proliferation.

Recent works suggest that YAP could be an oncogen, as YAP/TAZ mutations have been reported in certain types of cancer (breast, lung, ovary, colon…). In addition, some mutations in the Hippo/Yap pathway confer an overgrowth phenotype, visible on organ size. Finally, the dysregulation of the Hippo/yap pathway confers self-regenerative properties on cancer cells. (Cordenonsi. et al Cell 2011, Pan D. et al Genes & Dev. 2007, Pan D. et al Developmental Cell 2010)

Product Performance

1. Compatibility of the HTRF total and phospho-YAP(Ser127) cellular assays with different human and mouse cell lines

Human (HEK293, HEK293 A, U-2 OS, HeLA, MDA-MB-231, MCF 10A) and murine (NIH 3T3) cells were plated at 100,000 cells/ well in a 96 well plate in serum-deprived cell culture medium, and incubated for 24h at 37°C, 5% CO2. Medium was then removed and cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate was transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-YAP Ser127 or Total YAP detection reagents were added. The HTRF signal was recorded after an overnight incubation.

2. HTRF assay compared to Western Blot using phospho and total YAP cellular assays

Human MDA-MB-231 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48 h. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, then 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.

  • The HTRF phospho YAP assay is at least 4-fold more sensitive than the Western Blot. Using HTRF, 1,000 cells are sufficient for minimal signal detection while 4,000 cells are needed for a Western Blot signal.
  • The HTRF total YAP assay is 2-fold more sensitive than the Western Blot. Using HTRF , 2,000 cells are sufficient for minimal signal detection while 4,000 cells are needed for a Western Blot signal.

3. Serum dose-response measured by the HTRF phospho-YAP cellular assay

100,000 HEK 293A cells were plated in 96-well plate in serum-deprived cell culture medium, and incubated for 24h, at 37°C, 5% CO2. Increasing concentrations of serum were added for 1h. After cell culture medium removal, cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-YAP detection reagents were added. The HTRF signal was recorded after an overnight incubation. Stimulation with increasing concentrations of serum induces a decrease in YAP phosphorylation.

4. Kinetic of serum stimulation on phospho- and total YAP

100,000 of the HEK293A were plated in 96 well plate in serum-deprived cell culture medium, and incubated for 24h, at 37°C - 5% CO2. Serum was added for different times. After cell culture medium removal, cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho- or total YAP detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Maximum phosphorylation is obtained after a 1 hour stimulation with serum, while an overnight exposure to serum is needed to obtain the optimal quantity of the total YAP protein.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
YAP phospho-S127 kit - 500 tests64YAPPEG
YAP phospho-S127 kit - 10,000 tests64YAPPEH
YAP phospho-S127 kit - 50,000 tests64YAPPEY
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YAP total kit - 500 tests64YATPEG
YAP total kit - 10,000 tests64YATPEH
YAP total kit - 50,000 tests64YATPEY
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Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF
YAP phospho-S127 kit control lysate64YAPTDA
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YAP total kit control lysate64YATTDA
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