HTRF TOM20 Kit HTRF®

Associated products

The HTRF TOM20 kit is designed for the simple and rapid quantification of the protein in cell-based formats.

All inclusive kit
No-wash
Cell-based
Ease-of-use

Download the protocols

The HTRF TOM20 kit is designed for the simple and rapid quantification of the protein in cell-based formats.

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Overview

The removal of damaged mitochondria is critical for maintaining cellular homeostasis. The Translocase of Outer Membrane (TOM) receptor complex is responsible for the movement of proteins through the outer mitochondrial membrane of the mitochondria into the intermembrane space. TOM20 is a central component of the TOM complex. Monitoring the degradation of TOM20 is an indicator of mitophagy activation. This kit is intended to measure TOM20 in cellular lysates. The assay is a fast alternative to ELISA, thanks to the easy implementation of our Add and Read method.

Benefits

Validated on SH-SY5Y cell mitophagy model

Assay principle

TOM20 is measured using a sandwich immunoassay involving two specific anti-TOM20 antibodies, respectively labeled with HTRF donor and acceptor dyes. The intensity of the signal is directly proportional to the concentration of TOM20 present in the sample.

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 TOM20 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Kit characteristic

Analytical Performances
Time to results	ON at RT
LOD & LOQ (in Diluent)	25 pg/mL & 45 pg/mL
Range	45 – 5000 pg/mL

Precision

Intra-assay (n=24)

Sample	Mean [TOM20] (pg/mL)	CV
1	109	5.5 %
2	590	1.9 %
3	2048	2.1 %
	Mean CV	3.7 %

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Inter-assay (n=4)

Sample	TOM20 (pg/mL)	Mean (delta ratio)	CV
1	156	588	11 %
2	625	3604	5 %
3	2500	17989	9 %
			6 %

Each of the samples was measured in 4 different experiments, and the %CV for the concentration was calculated for each sample.

Valinomycin induced mitophagy on human SH-SY5Y cell line

Neuroblastoma SH-SY5Y (human) cells were plated (50,000 cells/well) in a 96 well plate and cultured overnight in complete culture medium(DMEM/F-12, 10mM HEPES + 10%SVF) at 37°C, 5% CO2.

The day after, medium was renewed and the cells were incubated for 24h at 37°, 5% CO2, with increasing concentrations of valinomycin, a mitochondrial uncoupler. After medium removal, the cells were lysed with 50 µL of 1X supplemented lysis buffer #4 for 30 min at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate followed by 4 µL of the HTRF TOM20 detection antibodies.The HTRF signal was recorded after an overnight incubation and the signals were interpolated on the TOM20 calibration curve built from serial dilution of TOM 20 standard.

An HTRF Ubiquitin Phospho-S65 assay was run on 14µL of lysates as a control for valinomycin mediated mitophagy activation.

TOM20 decrease upon valinomycin treatment on human SH-SY5Y cell line

Increase of phospho-(S65) Ubiquitin upon valinomycin treatment on human SH-SY5Y cell line

Simplified Pathway

In a depolarization-induced mitophagy process, PINK1 (PTEN-induced kinase 1) linked to the outer mitochondrial membrane is activated by dimerization and autophosphorylation. Once activated, PINK-1 phosphorylates Parkin, a Ubiquitin ligase, at position Ser65 of its Ubiquitin-like domain. PINK-1 also ubiquitinates proteins of the outer mitochondrial membrane and among them TOM 20 which once ubiquitinated is sent to proteasome. This is resulting to an increase of phospho-Ubiquitin concentration and a decrease of the TOM 20 concentration.