Total alpha Synuclein cellular kit
Simple, all-in-one kit for robust detection of total alpha Synuclein
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The HTRF TOM20 kit is designed for the simple and rapid quantification of the protein in cell-based formats.
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.
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.
Time to results | ON at RT |
LOD & LOQ (in Diluent) | 25 pg/mL & 45 pg/mL |
Range | 45 – 5000 pg/mL |
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.
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.
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.
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.
Product Insert TOM20 Kit / 64TOM20PEG-64TOM20PEH
64TOM20PEG-64TOM20PEH - Product Insert
Safety Data Sheet (DEU) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (ELL) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (ITA) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (SPA) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) TOM20 Kit / 64TOM20PEG
64TOM20PEG - Safety Data Sheet
Safety Data Sheet (DEU) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (ELL) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (ITA) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (SPA) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) TOM20 Kit / 64TOM20PEH
64TOM20PEH - Safety Data Sheet
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