Phospho-PDGFR beta (Tyr751) Cellular Assay Kit

Homogeneous cell-based assay for PDGF receptor β involved in angiogenesis and cancer 

Cisbio's cell-based homogeneous HTRF® phospho-PDGFRβ assay (Tyr751) enables rapid quantitative detection of the modulation of the platelet-derived growth factor receptor beta, phosphorylated on Tyrosine 751. PDGFRβ signals and crosstalks via both the PI3K/AKT and the Ras/Raf MAPK pathways. This cell surface tyrosine kinase receptor ensures the binding of particular growth factors, which trigger the MAPK pathway leading to cell proliferation, cell differentiation, and angiogenesis. When activated, PDGFRβ forms a dimer and becomes phosphorylated, which makes the phospho-PDGFRβ assay a valuable tool in oncology and metabolic disorder research. This simple add-and-read assay is ideal for screening small molecule compounds or biologics directly in cells. The phospho-PDGFRβ ready-to-use kit contains all the reagents you need and offers enhanced convenience over other immunoassay technologies.

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio's phospho-PDGFR-beta assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-PDGFR-beta antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-PDGFR-beta antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. 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 phospho-PDGFR-beta assay kit can be run with frozen cell lysates or fresh cells in culture. After cell lysis, phospho-PDGFR-beta can be quantitatively detected using the HTRF phospho-PDGFR-beta kit reagents and most TR-FRET multimode plate readers.

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 the detection of phosphorylated PDGFR-beta, lysates are subsequently transferred to a 384 small volume assay plate, where the HTRF reagents are added. This also enables the monitoring of cell 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.

Pathway

The Platelet-derived growth factor receptor (PDGF), is a transmembrane protein possessing an intracellular tyrosine kinase domain. PDGFRβ features two distinct polypeptide subunits, named α and β, both of which have tyrosine kinase activity.

Ligand binding induces receptor dimerization and auto-phosphorylation and results in both AKT/PI3K and MEK/ERK pathway activation. These pathways mediate survival, proliferation, growth, and differentiation.

Product performances

1 - Hepatic LX2 stellate cells stimulated with PDGF

Human PDGF was used to induce phosphorylation of PDGFRβ in human LX2 cells. LX2 cells, plated at different cell densities, were stimulated with various concentrations of human PDGF for 5 minutes. After a lysis step using 50 µL of supplemented lysis buffer, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-PDGFRβ detection reagents. The HTRF signal was recorded after 2h of incubation.

Phospho-PDGFRβ signal increases proportionally to the cellular density.

2 - Kinetics of PDGFRβ phosphorylation using mouse C2C12 cells

Human PDGF was used to induce phosphorylation of PDGFRβ in mouse C2C12 cells. The cells, plated at 50,000 cells per well, were stimulated with various concentrations of human PDGF for different times. After a lysis step using 50 µL of supplemented lysis buffer, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-PDGFRβ detection reagents. The HTRF signal was recorded after 2h of incubation.

Short optimization times in the range of minutes were sufficient to induce the receptor phosphorylation.

3 - Compatibility of the p-PDGFRβ assay with different cell lines

Human PDGF was used to induce phosphorylation of PDGFRβ in mouse C2C12 and NIH 3T3 cells, as well as in human LX2 cells. The cells, plated at 100,000 cells per well, were stimulated with 10ng/mL of human PDGF for 5 minutes. After a lysis step using 50 µL of supplemented lysis buffer, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-PDGFRβ detection reagents. The HTRF signal was recorded after 2h of incubation.

4 - HTRF phospho-PDGFRβ cellular assay compared to Western Blot

C2C12 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. The cells were then stimulated with PDGF for 5 min. Following medium removal, the cells were lysed using 3mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution was dispensed and analyzed side-by-side by Western Blot and by HTRF.

Using HTRF® Phospho-PDGFRβ, fewer than 1,250 cells were sufficient for minimal signal detection, while 20,000 cells were needed for a Western Blot signal. The HTRF® assay is 16-fold more sensitive than the Western Blot.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
PDGFR beta phospho-Y751 kit - 500 tests64PDGPEG
PDGFR beta phospho-Y751 kit - 10,000 tests64PDGPEH
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PDGFR beta phospho-Y751 kit - 50,000 tests64PDGPEY
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Companion products

DescriptionCat. noProduct insertMSDS
PDGFR beta phospho-Y751 kit - control lysate64PDGTDA
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