Revvity, 64P62S4PEH, HTRF Human and Mouse Phospho-p62 (SQSTM1) (Ser403) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately quantifies phosphorylated p62/SQSTM1 at Ser403. p62/SQSTM1 is a ubiquitin-binding protein Involved in the regulation of autophagy. This is a cellular mechanism which enables the clearance of abnormal proteins or organelles, and it plays a role in cancer, neurodegenerative diseases, as well as cardiovascular and infectious diseases.
p62/SQSTM1 functions as a bridge between polyubiquitinated cargo protein and autophagosomes by interacting directly with both the cargo protein and a protein called LC3, which is involved in autophagosome formation. Recently, p62/SQSTM1 activation has been associated with ULK1 and mTor- induced phosphorylation on different residues, like Ser 403.
The activated autophagy pathway promotes the degradation of p62-associated ubiquitinated protein cargoes.
How it works
Phospho-p62/SQSTM1 (Ser403) assay principle
Revvity's Phospho-p62/SQSTM1 (Ser403) assay measures p62 when phosphorylated at Ser403. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-p62 (Ser403) assay uses 2 labeled antibodies, one with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune complex formation involving the two labeled antibodies, which brings the donor fluorophore into close proximity to the acceptor and thereby generates 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-p62/SQSTM1 (Ser403) 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 Phospho-p62/SQSTM1 (Ser403) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-p62/SQSTM1 (Ser403) one-plate assay protocol
Detection of Phosphorylated p62/SQSTM1 (Ser 403) 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-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-Cardiovascular Infectious Diseases Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points