This HTRF kit enables the cell-based quantitative detection of total PINK1 as a readout of the Mitophagy pathway.
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).
The Total PINK1 assay is designed to monitor the cellular level of PINK1 protein.
PINK1 is a ser/thr kinase which plays a key role in PINK1-Parkin-mediated mitophagy. This signaling pathway is essential for toxic mitochondrial products removal maintaining cellular homeostasis. Dysregulation of this signaling is linked to neurodegenerative diseases, mainly Parkinson's disease.
In damaged depolarized mitochondria, PINK1 accumulates on the mitochondria outer membrane and activates PINK1-Parkin pathway by phosphorylating two substrates, the Ubiquitin-like domain of Parkin and Ubiquitin chains at position Ser65. Parkin is a Ubiquitin ligase E3 that conjugates Ubiquitin to impaired-mitochondrial proteins, leading to organelle degradation. The amplified PINK1/Parkin dependent Ubiquitin functions act as a signal for the sequestration and degradation of damaged mitochondria (mitophagy).
Depolarization of mitochondria by uncoupling agents, such as CCCP and valinomycin, causes PINK1 to accumulate on the outer mitochondrial membrane and trigger PhosphoS65-Ubiquitin production.
Application |
Cell Signaling
|
---|---|
Brand |
HTRF
|
Detection Modality |
HTRF
|
Molecular Modification |
Total
|
Product Group |
Kit
|
Sample Volume |
16 µL
|
Shipping Conditions |
Shipped in Dry Ice
|
Target Class |
Phosphoproteins
|
Technology |
TR-FRET
|
Unit Size |
500 Assay Points
|
The HTRF Total PINK1 assay quantifies the expression level of PINK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total PINK1 assay uses two labeled antibodies, one coupled to a donor fluorophore, and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of PINK1 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.
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 PINK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Total PINK1 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.
SH-SY5Y (neuroblastoma) or PC3 (prostatic adenocarcinoma) cells were cultured in a 96-well plate at a density of 200,000 cells/well for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), a mitochondrial uncoupler. After 6h of incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#1 (1X) were dispensed into each well. Following cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total PINK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.
HTRF Alpha-Tubulin Housekeeping assay (64ATUBPEG/H) was run simultaneously on 4µL of 1/5 diluted lysates as a normalization tool. The HTRF signal was recorded after an overnight incubation.
In both cell types, the CCCP compound triggered a dose-dependent increase in the PINK1 protein level. These results suggest that CCCP induces PINK1 accumulation, with the expected EC50 in the low µM range.
SH-SY5Y (neuroblastoma) or PC3 (prostatic adenocarcinoma) cells were cultured in a 96-well plate at a density of 200,000 cells /well for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Valinomycin, a mitochondrial uncoupler. After 4h of incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#1 (1X) were dispensed into each well. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total PINK1 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.
HTRF Alpha-Tubulin Housekeeping assay (64ATUBPEG/H) was run simultaneously on 4µL of 1/5 diluted lysates as a normalization tool. The HTRF signal was recorded after an overnight incubation.
In both cell types, the Valinomycin compound triggered a dose-dependent increase in the PINK1 protein level. These results suggest that Valinomycin induces PINK1 accumulation , with the expected EC50 in the nM range.
Discover the versatility and precision of Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our HTRF portfolio offers a...
This guide provides you an overview of HTRF applications in several therapeutic areas.
This document includes detailed tables listing HTRF™, AlphaLISA™ SureFire® Ultra™, and Alpha SureFire® Ultra™ Multiplex assays...
Are you looking for technical documents related to the product? We have categorized them in dedicated sections below. Explore now.
We are here to answer your questions.