Advanced phospho-ERK (Thr202/Tyr204) cellular kit
Simple, all-in-one kit for robust detection of Phospho-ERK.
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This HTRF kit enables the cell-based quantitative detection of phosphorylated RET on Tyrosines as a readout of neurotrophic factor activation.
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.
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.
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.
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.
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.
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.
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.
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 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.
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
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 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
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
Safety Data Sheet (DEU) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (ELL) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (ITA) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (SPA) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) RET p-pan Y Kit / 64RETYPEG
64RETYPEG - Safety Data Sheet
Safety Data Sheet (DEU) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (ELL) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (ITA) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (SPA) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) RET p-pan Y Kit / 64RETYPEH
64RETYPEH - Safety Data Sheet
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