Revvity, 64IRTPEH, HTRF Human and Mouse Total IRS2 Detection Kit, 10,000 Assay Points

Overview
Insulin receptor substrates (IRS) 1 and 2 are the primary substrates of Insulin/IGF1 receptors, and mediate cell growth/survival and metabolic homeostasis. While IRS1 is primarily found in adipose tissue, IRS2 is the predominant form in pancreatic-beta cells and the central nervous system (CNS). Decreased IRS2 levels are associated with diabetes and neurodegeneration, whereas highly overexpressed levels of IRS2 drive tumor motility and invasion. This cell-based assay measures the modulation of IRS2 protein levels.
How it works
Total IRS2 assay principle
The total IRS2 assay quantifies the expression level of IRS2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-IRS2 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 IRS2 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 proteins expression under a no-wash assay format.

 

Total IRS2 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding total IRS2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total IRS2 1-plate assay protocol
Detection of total IRS2 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-Metabolism/Diabetes Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points