Revvity, 64NH2PEH, HTRF Human and Mouse Total SHP2 Detection Kit, 10,000 Assay Points

Overview
The Total SHP2 cellular assay monitors the expression level of SHP2, and can be used as a normalization assay with our phospho-SHP2 Y542 kit. Because these phospho and total SHP2 assays are compatible, the two kits can be used in parallel on the same lysates.
Many cancer cells overexpress checkpoint inhibitor ligands such as PD-L1. PD-L1 binds its counterpart checkpoint inhibitor receptor PD1, present at the surface of T lymphocytes. In turn, the PD1-PDL1 complex recruits and activates inhibitory effectors, such as SHP1 or SHP2. These two phosphatases, which are phosphorylated on Tyr564 and Tyr542 respectively by the kinase Lck, trigger the dephosphorylation of signaling proteins such as ZAP-70 or SLP-76, involved in the T cell activation pathway. Finally, activated SHP1 and SHP2 participate in T cell inactivation.
Preventing the activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.
How it works
Total SHP2 assay principle
The Total SHP2 assay measures the expression level of SHP2 independently of its phosphorylation state. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total SHP2 assay uses 2 labeled antibodies, one with a donor fluorophore, the other with an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SHP2 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 SHP2 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 Total SHP2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total SHP2 one-plate assay protocol
Detection of Total SHP2 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 2 Lysis Buffer 3
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-Infectious Diseases Oncology & Inflammation
Unit Size-10,000 assay points