Revvity, 64PINKTPEG, HTRF Human Total PINK1 Detection Kit, 500 Assay Points

Overview
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.
How it works
Total PINK1 assay principle
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.

 

Total PINK1 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 PINK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total PINK1 one-plate assay protocol
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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-500 assay points