Total VASP cellular kit HTRF®

Associated products

The total VASP kit detects cellular VASP and can be used as a normalization assay for phospho-VASP kits to help provide optimal readout of PKA and PKG activation.

Ready-to-use
High sensitivity
Faster and more convenient than ELISA

Download the protocols

The total VASP kit detects cellular VASP and can be used as a normalization assay for phospho-VASP kits to help provide optimal readout of PKA and PKG activation.

Size

Cat. number selected :

Quantity

Overview

Total VASP cell-based assay is designed to monitor the expression level of VASP proteins, both phosphorylated and unphosphorylated. It is compatible with our Phospho-VASP kits, and enables the analysis of phosphorylated and total proteins from a single sample for better readout of PKA and PKG activation. VASP is involved in cell motility, migration, and adhesion. This makes phospho-VASP assaying a valuable tool in cardiovascular, oncology, and inflammation research.

Benefits

SPECTIFICITY
PRECISION
DATA NORMALIZATION

Total-VASP assay principle

The Total-VASP assay quantifies the expression level of VASP in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-VASP 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 VASP 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-VASP 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-VASP HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-VASP 1-plate assay protocol

Detection of total VASP 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.

HTRF assay compared to Western Blot using total VASP 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 total VASP 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 20 000 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.

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 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.

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.

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

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

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

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

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

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

Product Insert VASP total Kit / 63ADK067PEG-63ADK067PEH

63ADK067PEG-63ADK067PEH - Product Insert

HTRF Product Catalog

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

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

Safety Data Sheet (DEU) VASP total Kit / 63ADK067PEG