Phospho-YAP (Ser127) cellular kit
All-in-one kit for robust detection of Phospho-YAP (Ser127)
Total YAP cellular kit HTRF®
The total YAP kit detects cellular YAP and can be combined to our phospho-YAP kit for an optimal readout of the Hippo-YAP pathway
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Ready-to-use
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High sensitivity
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Faster and more convenient than ELISA
Overview
The Total YAP cell-based assay is designed to monitor the expression level of YAP proteins, both phosphorylated and unphosphorylated. It uses the same buffers as our Phospho-YAP kit, and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of the Hippo-YAP pathway. With its central mediator role in the Hippo Pathway, YAP is an important target for oncological disease studies.
Benefits
- SPECIFICITY
- PRECISION
- DATA NORMALIZATION
Total-YAP assay principle
The Total-YAP assay quantifies the expression level of YAP in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-YAP assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of YAP in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.
Total-YAP 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total-YAP HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-YAP 1-plate assay protocol
Detection of total YAP 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 enables miniaturization while maintaining robust HTRF quality.
Compatibility of the HTRF Total YAP assay with various cell lines
Human (HEK293, HEK293 A, U-2 OS, HeLA, breast cancer cells, 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 Total YAP Ser127 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
HTRF assay vs WB using total YAP cellular assay
Human breast cancer 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 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.
Kinetic of serum stimulation on 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 total YAP detection reagents were added. The HTRF signal was recorded after an overnight incubation. Overnight exposure to serum is needed to obtain the optimal quantity of the total YAP protein.
Hippo-YAP cell signaling Active Pathway
The Hippo/YAP pathway regulates organ size by playing a role in the balance between proliferation & apoptosis. 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 activates MTS kinase, 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. YAP/TAZ mutations have been reported in certain types of cancer (e.g. breast, lung, ovary, and colon).
Hippo-YAP cell signaling Inactive Pathway
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. When the Hippo/YAP pathway is inactivated, YAP/TAZ accumulates in the nucleus and induces the transcription of genes involved in cell proliferation. In addition to its properties and oncogen potential, 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)
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Open R&D: Sanofi Access Platform
In collaboration with Sanofi - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Insight into fibrosis development and signaling pathways
A comprehensive overview of fibrosis development - Guides
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Manual YAP total Kit / 64YATPEG-64YATPEH
64YATPEG-64YATPEH - Product Insert
Safety Data Sheet (DEU) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (ELL) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (ITA) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (SPA) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) YAP total Kit / 64YATPEG
64YATPEG - Safety Data Sheet
Safety Data Sheet (ELL) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (DEU) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (ITA) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (SPA) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) YAP total Kit / 64YATPEH
64YATPEH - Safety Data Sheet
Safety Data Sheet (DEU) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (ELL) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (ITA) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (SPA) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Safety Data Sheet (ENG-US) YAP total Kit / 64YATPEY
64YATPEY - Safety Data Sheet
Plate Reader Requirement
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