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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  • Faster and more convenient than ELISA 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.

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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 IRAK3 Assay principle

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 IRAK3 2-plate Assay protocol

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.
IRS2 upregulation in INS-1E beta cells using glucose
Upregulation of IRS2 protein levels in INS-1E rat pancreatic ß-cells using glucose and forskolin

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.
Upregulation of IRS2 in HEK293 cells using forskolin

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 SH-SY5Y human neuroblastoma cell line

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

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.
IRS2 signaling pathway in pancreatic beta cells
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Cisbio lysis buffer compatibility

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Species compatibility

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Product Insert IRS2 total Kit / 64IRTPEG-64IRTPEH

64IRTPEG-64IRTPEH - Product Insert

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