Revvity, 64UBIS65PEG, HTRF Human and Mouse Phospho-Ubiquitin (Ser65) Detection Kit, 500 Assay Points

Overview
The Phospho-Ubiquitin (Ser65) Cellular assay is designed to monitor the level of the phosphorylated form of the Ubiquitin chain. Ubiquitin itself is a 76 amino acid protein (8.5 kDa) that is ubiquitously expressed and highly conserved, with 100% AA sequence identity between human and mouse.
Phosphorylation of Ubiquitin is a key step in the process of mitochondria quality control through the Parkin/PINK-1 interaction. Parkin is a Ubiquitin ligase E3 that conjugates Ubiquitin to impaired-mitochondrial proteins, leading to organelle degradation. PINK1 is a Ser/Thr kinase that accumulates only on impaired mitochondria and phosphorylates two substrates, the Ubiquitin-like domain of Parkin and Ubiquitin chains.
The amplified Parkin/PINK-1 dependent Ubiquitin functions act as a signal for the sequestration and degradation of damaged mitochondria (mitophagy). Dysregulation of this signaling is linked to some forms of Parkinson's Disease.
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
Phospho-Ubiquitin (Ser65) assay principle
The Phospho-Ubiquitin (Ser65) assay measures Ubiquitin when phosphorylated at Ser65. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-Ubiquitin (Ser65) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Activation Buffer followed by Phospho-Ubiquitin (Ser65) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-Ubiquitin (Ser65) one-plate assay protocol
Detection of Phosphorylated Ubiquitin (Ser65) 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 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Neuroscience
Unit Size-500 assay points