Phospho-VASP (Ser157), Phospho-VASP (Ser239) & Total VASP Cellular Assay Kits

Phospho- and total VASP assays for monitoring PKA and PKG activation

Cisbio's cell-based homogeneous HTRF® phospho-VASP assays enable quantitative detection of the modulation of VASP phosphorylated on Serine 157 or Serine 239. In endothelial cells, VASP is phosphorylated by the PKA kinase on Serine 157 and by PKG kinase on Serine 239. It  is involved in cell motility, migration, and adhesion. This makes phospho-VASP assays a valuable tool in cardiovascular, oncology and inflammation research.

Using a streamlined protocol without any washing steps and amenable to low-volume formats, the phospho-VASP assay can be used from basic research up to preclinical drug discovery phases. The kit contains all the reagents you need, and offers increased throughput compared to ELISA or Western Blot.

 

Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total VASP assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-VASP antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with VASP, and the proximity of donor and acceptor then leads 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-VASP can be quantitatively detected using the HTRF phospho-VASP (Ser157), phospho-VASP (Ser239) and total VASP 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 384-well sv assay plate, where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

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.

Product performances

1. HTRF assay compared to Western Blot using phospho & total VASP cellular assays 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 or phospho-VASP (Ser239) detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.

For both HTRF assays (phospho- and total), just 2 500 cells were sufficient for minimal signal detection, while from 20 000 up to 40 000 cells were needed for a Western Blot signal detection, showing that the VASP HTRF® assays are at least 4-fold more sensitive than the Western Blot.

2. Validation on A431 and NIH3T3 cells 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.

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.

Simplified pathway

Function and regulation of VASP (Vasodilator stimulated phosphoprotein)

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
VASP phospho-S239 kit - 500 tests63ADK065PEG
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VASP phospho-S239 kit - 10,000 tests63ADK065PEH
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VASP phospho-S239 kit - 50,000 tests63ADK065PEY
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VASP phospho-S157 kit - 500 tests63ADK066PEG
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VASP phospho-S157 kit - 10,000 tests63ADK066PEH
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VASP phospho-S157 kit - 50,000 tests63ADK066PEY
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VASP total kit - 500 tests63ADK067PEG
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VASP total kit - 10,000 tests63ADK067PEH
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VASP total kit - 50,000 tests63ADK067PEY
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Companion products

DescriptionCat. noProduct insertMSDS
VASP phospho-S239 kit control lysate63ADK065TDA
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VASP phospho-S157 kit control lysate63ADK066TDA
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VASP total kit control lysate63ADK067TDA
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