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HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
Generic HTRF biomarker image
| Feature | Specification |
|---|---|
| Application | Protein Quantification |
| Sample Volume | 16 µL |
Product variants
Unit Size: 500 assay points
Part #:
64TOM20PEG
Unit Size: 10,000 assay points
Part #:
64TOM20PEH
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
Generic HTRF biomarker image
HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
Product information
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.
How it works
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.
Analytical performance
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.
Assay validation
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.
Simplified pathway
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 phosphorylates ubiquitinated proteins of the outer mitochondrial membrane and among them TOM20 which once ubiquitinated is sent to proteasome. This is resulting to an increase of phospho-Ubiquitin concentration and a decrease of the TOM20 concentration.
Specifications
| Application |
Protein Quantification
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 1
Lysis Buffer 4
|
| Product Group |
Kit
|
| Sample Volume |
16 µL
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Target Class |
Biomarkers
|
| Target Species |
Human
Mouse
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Oncology & Inflammation
|
| Unit Size |
500 assay points
|
Video gallery
HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
HTRF Human and Mouse TOM20 Detection Kit, 500 Assay Points
Resources
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