The total IRS2 assay enables the detection of cellular IRS2 and can be used as a normalization assay for the phospho-IRS2 kit.
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  • Ready-to-use
  • High sensitivity
  • Faster and more convenient than ELISA
The total IRS2 assay enables the detection of cellular IRS2 and can be used as a normalization assay for the phospho-IRS2 kit.
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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.

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 protein’s 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.

IRS2 upregulation in INS-1E beta rat cells using glucose and forskolin

INS-1E cells were plated in a 24-well plate and cultured for 4 days. A starvation step using Krebs Ringer Buffer (KRB) without glucose was performed for 1 hour before stimulating the cells with glucose +/- forskolin (diluted in culture medium without glucose). After 6 hours of treatment, the medium was removed and cells were lysed and transferred into a 384-well low volume white microplate with HTRF total IRS2 detection reagents added. The HTRF signal was recorded after an overnight incubation.

Upregulation of IRS2 protein levels in HEK293 human embryonic kidney cells using forskolin

HEK293 cells were plated at 100,000 cells/well in a 96-well plate, and incubated for 24 hours at 37°C, 5% CO2. After treatment for 30 minutess or 4 hours with 1 nM forskolin, the medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were then transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Detection of IRS2 in SH-SY5Y human neuroblastoma cell line

SH-SY5Y cells were plated in a 6-well plate at two different cell densities (2 million and 4 million cells/well) and incubated for 24 hours at 37°C, 5% CO2. The medium was then removed and cells were lysed with 200 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Detection of IRS2 in MCF-7 human breast cancer cell line

MCF-7 cells were plated in a 96-well plate at 100,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. The medium was then removed and cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutess at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF total IRS2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Function and regulation of IRS2 in pancreatic ß-cells

IRS2 expression in pancreatic-beta cells by both 2nd messengers: glucose and GLP-1. These promote activation of transcription factor CERB leading to increased IRS2 expression. IRS2 is crucial to functional Insulin/IGF-1 signaling, promotiing beta-cell growth, survival and function. Pro-inflammatory adipocytokines, free fatty acids and cellular stress promote IRS2 degradation and contribute to spontaneous beta-cell apoptosis, decreased beta-cell mass, insulin deficiency and eventually metabolic disorders such as diabetes.
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