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HTRF Phospho-RET (pan) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated RET on Tyrosines as a readout of neurotrophic factor activation.


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  • No-wash No-wash
  • High sensitivity High sensitivity
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption

This HTRF kit enables the cell-based quantitative detection of phosphorylated RET on Tyrosines as a readout of neurotrophic factor activation.


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Overview

The HTRF Phospho-RET (pan) cell-based assay conveniently and accurately detects phosphorylated RET on Tyrosine residues. ​​​​​​​The assay measures both short and long RET isoforms.

The proto-oncogene RET (Rearranged during Transfection), also known as c-Ret, is a receptor tyrosine kinase primarily expressed as two isoforms of 1072 & 1114 amino acids. RET activation by Glial cell line-derived neurotrophic Family Ligands (GFL) leads to its dimerization and autophosphorylation on intracellular Tyrosine residues. RET is required for the development of the nervous system and several other tissues. RET alterations have been detected in numerous human cancers. The development of highly selective and potent RET kinase inhibitors is therefore of great interest to treat RET-altered cancers. Recently, RET has also been linked to neurodegeneration.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-RET (pan) assay principle

The Phospho-RET (pan) assay measures RET when phosphorylated on Tyrosines. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to phosphorylated tyrosine residues, the second for its ability to recognize specifically the protein independently 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.

Principle of the HTRF phospho-RET (pan) assay

Phospho-RET (pan) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-RET (pan) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF phospho-RET (pan Tyr) assay

Phospho-RET (pan) one-plate assay protocol

Detection of Phosphorylated RET (pan) 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.

One-plate protocol of the HTRF phospho-RET (pan Tyr) assay

Induction of RET phosphorylation in mouse neuroblastoma Neuro-2a cells

The mouse neuroblastoma cell line Neuro-2a was seeded in a 96-well culture-treated plate under 50,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 15 minutes with increasing concentrations of Pervanadate, in absence or in presence of mouse GDNF at 100 ng/mL. After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

GDNF-induced RET phosphorylation is visible in absence of Pervanadate, and the detection is improved in presence of 6.25 µM and 12.5 µM Pervanadate (prevention of tyrosine dephosphorylation). The highest level of Phospho-RET is obtained with 50 µM Pervanadate alone.

The total RET assay shows a constant level of Total RET in all experimental conditions, confirming that high doses of Pervanadate did not induce cell detachment.

Phospho-RET (pan) in Neuro-2a cells treated with GDNF and Pervanadate
Total RET in Neuro-2a cells treated with GDNF and Pervanadate

Inhibition of RET phosphorylation in mouse neuroblastoma Neuro-2a cells

The mouse neuroblastoma cell line Neuro-2a was seeded in a 96-well culture-treated plate under 100,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 2 hours with increasing doses of Pralsetinib, and 100 µM Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the RET kinase inhibitor Pralsetinib induced a dose-dependent decrease in RET phosphorylation, without effect on the expression level of the receptor.

Inhibition of RET in Neuro-2a cells using Pralsetinib

Inhibition of RET phosphorylation in human neuroblastoma SH-SY5Y cells

The human neuroblastoma cell line SH-SY5Y was seeded in a 96-well culture-treated plate under 100,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 2 hours with increasing doses of Pralsetinib and Selpercatinib, and 100 µM Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, both RET kinase inhibitors induced a dose-dependent decrease in RET phosphorylation, without effect on the expression level of the receptor.

RET inhibition in human SH-SY5Y cells treated with Pralsetinib
RET inhibition in human SH-SY5Y cells treated with Selpercatinib

Inhibition of RET phosphorylation in the human lung adenocarcinoma cell line LC-2/ad

The human lung adenocarcinoma cell line LC-2/ad was seeded in a 96-well culture-treated plate under 50,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were treated for 2 hours with increasing doses of Pralsetinib, and 100 µM Pervanadate were added 30 minutes before the end of the treatment. The cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-RET (pan) or Total-RET detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, the RET kinase inhibitor Pralsetinib induced a dose-dependent decrease in RET phosphorylation, without significant effect on the expression level of the receptor.

RET inhibition in human LC-2/ad cells treated with Pralsetinib

RET Signaling Pathway

RET is activated by Glial cell line-derived neurotrophic Family Ligands (GFL) including GDNF (glial cell line-derived neurotrophic factor), NRTN (neurturin), ARTN (artemin) and PSPN (persephin). Ligand binding to GDNF Family Receptor-α co-receptors (GFRα1/2/3/4) triggers the formation of a complex with RET, leading to RET dimerization and autophosphorylation on multiple intracellular tyrosines. Phosphorylated tyrosine residues serve as docking sites for various adaptor proteins that induce the activation of downstream signaling pathways (MAPK, PI3K/AKT, STAT3...) necessary for cell survival, differentiation, proliferation and motility.

RET Signaling Pathway

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - 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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

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

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

Product Insert RET p-pan Y Kit / 64RETYPEG-64RETYPEH

64RETYPEG-64RETYPEH - Product Insert

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

Plate Reader Requirement

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