Phospho-PDGFR beta (Tyr751) cellular kit

The phospho-PDGFR beta (Tyr751) kit is designed for the cell-based quantitative detection of PDGFR beta phosphorylation.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The phospho-PDGFR beta (Tyr751) kit is designed for the cell-based quantitative detection of PDGFR beta phosphorylation.
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Overview

The Phospho-PDGFRß (Tyr751) assay enables the quantitative detection of the modulation of the platelet-derived growth factor receptor beta, phosphorylated on Tyrosine 751. PDGFR beta 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, PDGFRbeta forms a dimer and becomes phosphorylated, which means the phospho-PDGFRß assay is a valuable tool in oncology research.

Benefits

  • COMPATIBLE WITH HUMAN & RODENT CELL
  • PRECISION
  • SPECIFICITY

Phospho-PDGFRß (Tyr751) assay principle

The Phospho-PDGFRß (Tyr751) assay measures PDGFRß when phosphorylated at Tyr751. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-PDGFRß (Tyr751) 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-PDGFRß (Tyr751) assay principle

Phospho-PDGFRß (Tyr751) 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-PDGFRß (Tyr751) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-PDGFRß (Tyr751) 2-plate assay protocol

Phospho-PDGFRß (Tyr751) 1-plate assay protocol

Detection of Phosphorylated PDGFRß (Tyr751) 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-PDGFRß (Tyr751) 1-plate assay protocol

Hepatic LX2 stellate cells stimulated with PDGF

Human PDGF was used to induce phosphorylation of PDGFRß 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-PDGFRß detection reagents. The HTRF signal was recorded after 2h of incubation. Phospho-PDGFRß signal increases proportionally to the cellular density.
HTRF phospho-PDGFRb measurement after induction by Human PDGF in LX2 stellate cells

Kinetics of PDGFRß phosphorylation using mouse C2C12 cells

Human PDGF was used to induce phosphorylation of PDGFRß 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-PDGFRß 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.
HTRF phospho-PDGFRb measurement after induction by Human PDGF in C2C12 cells

Compatibility of the p-PDGFRß assay with different cell lines

Human PDGF was used to induce phosphorylation of PDGFRß 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-PDGFRß detection reagents. The HTRF signal was recorded after 2h of incubation.
Phospho-PDGFRb measurement on various cell lines

HTRF phospho-PDGFRß 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-PDGFRß, 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.

Phospho-PDGFR beta simplified pathway

The Platelet-derived growth factor receptor (PDGF), is a transmembrane protein possessing an intracellular tyrosine kinase domain. PDGFRß features two distinct polypeptide subunits, named a and ß, 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.
Phospho-PDGFR beta signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

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

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

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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