The phospho-VASP (Ser239) enables the cell-based quantitative detection of VASP phosphorylated on Ser 239 for monitoring PKA and PKG activation.
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  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The phospho-VASP (Ser239) enables the cell-based quantitative detection of VASP phosphorylated on Ser 239 for monitoring PKA and PKG activation.
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Overview

Phospho-VASP (Ser239) cellular assay is ideal for monitoring PKG activation, which phosphorylates VASP on Serine 239. VASP is involved in cell motility, migration, and adhesion. This makes phospho-VASP assaying a valuable tool in cardiovascular, oncology, and inflammation research.

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Phospho-VASP (Ser239) assay principle

The Phospho-VASP (Ser239) assay measures VASP when phosphorylated at Ser239. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-VASP (Ser239) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables 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-VASP (Ser239) assay principle

Phospho-VASP (Ser239) 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 phospho-VASP (Ser239) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-VASP (Ser239) Cellular Assay 2-plate assay protocol

Phospho-VASP (Ser239) 1-plate assay protocol

Detection of Phosphorylated VASP (Ser239) 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.
Phospho-VASP (Ser239) Cellular Assay 1-plate protocol

HTRF assay compared to Western Blot using phospho-VASP (s239) assay on mouse NIH3T3 cells

Mouse NIH3T3 cells were plated at 8.106 cells in a T175 cm2 flask and incubated for 48h at 37°C, 5% CO2. After 50µM forskolin treatment for 30 minutes, the cells were lysed with 3mL of lysis buffer for 30min at RT under gentle shaking. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-VASP (Ser239) detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF. For HTRF assays, 2 500 cells were enough for minimal signal detection, while 40 000 cells were needed for a Western Blot signal detection. Thus, phospho-VASP (Ser239) HTRF assays is at least 4-fold more sensitive than the Western Blot.
HTRF assay compared to Western Blot using phospho-VASP (s239) assay on mouse NIH3T3 cells

Validation on A431cells treated with Forskolin

Human A431 cells were plated at 100,000 cells/well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 30 min with increasing concentrations of Forskolin, the medium was removed and the cells were lysed with 60 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-VASP (Ser157), phosphor-VASP (S239) or total VASP detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Validation on A431 cells treated with Forskolin

Validation on NIH3T3 cells treated with Forskolin

Mouse NIH3T3 cells were plated at 100,000 cells/well in a 96 well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 30 min with increasing concentrations of Forskolin, the medium was removed and the cells were lysed with 60 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-VASP (Ser157), phosphor-VASP (S239) or total VASP detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Validation on NIH3T3 cells treated with Forskolin

Function and regulation of VASP

Vasodilator-stimulated phosphoprotein (VASP) is an actin-associated protein and a member of the Ena-VASP family. VASP stimulates actin filament elongation, and is involved in cytoskeleton remodeling and cell polarity. VASP proteins are involved in axon guidance, platelet activation and cell migration. In platelets, VASP is a major substrate for cAMP-dependent protein kinase A (PKA) and cGMP-dependent protein kinase G (PKG). Whereas the preferred site for PKA is Ser-157, the preferred site for PKG is Ser-239. Phosphorylation modulates F-actin binding, actin filament elongation and platelet activation. VASP phosphorylation is often used to monitor the effect of drugs that reduce platelet reactivity in cardiovascular diseases.
Pathway of Vasodilator-stimulated phosphoprotein
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

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Cisbio starter packs

96-tests kits - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

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

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

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

Product Insert VASP P-S239 Kit / 63ADK065PEG-63ADK065PEH

63ADK065PEG-63ADK065PEH - Product Insert

Cisbio Product Catalog 2019

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

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