Revvity, 64N62PEG, HTRF Human and Mouse Total p62 (SQSTM1) Detection Kit, 500 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately monitors the expression level of p62/SQSTM1 proteins, both phosphorylated and unphosphorylated. It uses the same buffers as our Phospho-p62/SQSTM1 kit, and enables the analysis of phosphorylated and total proteins from a single sample as a pivotal readout of the autophagy pathway. Autophagy is a cellular mechanism which enables the clearance of abnormal proteins or organelles, and plays a role in cancer, neurodegenerative diseases, as well as cardiovascular and infectious diseases.
p62/SQSTM1 is a ubiquitin-binding protein and functions as a bridge between polyubiquitinated cargo protein and autophagosomes. p62 interacts directly with both the cargo protein and a protein called LC3 which is involved in the autophagosome formation. Finally, the activated autophagy pathway promotes the degradation of p62-associated ubiquitinated protein cargoes.
How it works
Total p62/SQSTM1 assay principle
The Total-p62/SQSTM1 assay quantifies the expression level of p62 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total-p62/SQSTM1 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of p62/SQSTM1 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-p62/SQSTM1 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total-p62/SQSTM1 detection reagents. This protocol enables the cells' viability and confluence to be monitored

 

Total-p62/SQSTM1 (Ser403) one-plate assay protocol
Detection of Total p62/SQSTM1 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 Lysis Buffer 5
Molecular Modification-Total
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-Cardiovascular Infectious Diseases Neuroscience Oncology & Inflammation
Unit Size-500 assay points