Total-RET assay principle

The HTRF Total-RET assay quantifies the expression level of RET in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-RET assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of RET in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-RET 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 Total-RET HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-RET one-plate assay protocol

Detection of Total-RET 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.

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 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.

HTRF Total-RET assay compared to Western Blot

SH-SY5Y cells were cultured in a T175 flask in complete culture medium for 48h at 37°C, 5% CO2. The cells were treated with 100 µM Pervanadate for 30 minutes and lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total RET detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF total RET assay, 3,400 cells/well were enough to detect a significant signal, while 27,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF total RET assay was 8 times more sensitive than the Western Blot technique.

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.