Total IRS2 Cellular Assay Kit

Total IRS2 assay to study pancreatic β-cell growth/survival and central nutrient sensing

Insulin Receptor Substrates 1 and 2 (IRS1/2) are the primary substrates of Insulin/IGF1 receptors, and mediate the activation of the downstream signaling pathways AKT and ERK. These lead to cell growth/survival and metabolic homeostasis.

While IRS1 is most important in adipose tissue and skeletal muscles, IRS2 is the predominant form in pancreatic β-cells and in the central nervous system (CNS).

Decreased IRS2 protein levels are associated with metabolic disorders (such as diabetes) and neurodegeneration. Conversely, IRS2 overexpression drives tumor motility and invasion.

This cell-based assay is designed to monitor the modulation of IRS2 protein levels.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

The assay can be run with cell lysates or using whole cells.

Cisbio Bioassay’s total IRS2 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-IRS2 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with IRS2, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assay can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous total IRS2 can be quantitatively detected using the HTRF total IRS2 cellular assay kit reagents and most TR-FRET multimode plate readers.

The assay can be run under a two-plate protocol. It can also be further streamlined to a one-step assay.

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred into a 384-well low volume white microplate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

Detection of IRS2 with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.

Simplified Pathway

Function and regulation of IRS2 in pancreatic β-cells

IRS2 expression in pancreatic β-cells is stimulated by glucose and GLP-1: glucose induces ATP production and Ca2+ entry into the cell, while GLP-1 triggers the production of cAMP. Both 2nd messengers promote the activation of the transcription factor CREB, leading to increased IRS2 expression.

IRS2 is essential for maintaining functional IGF-1/Insulin signaling, which leads to the activation of AKT and ERK pathways and promotes β-cell growth, survival and function.                                 

Pro-inflammatory adipocytokines (such as TNF-α, IL-6, and IL-1β), free fatty acids and cellular stress promote the activation of the stress kinases IKKβ, JNK, PKC-δ and SOCS-1/3, which increase the proteasomal degradation of IRS2 and therefore inhibit the IGF-1/Insulin signaling pathway. 

Decreased IRS2 protein levels cause spontaneous β-cell apoptosis, leading to reduced β-cell mass, insulin deficiency, and finally to metabolic disorders such as diabetes.

Product Performance

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

INS-1E cells were plated in a 24-well plate (400,000 cells/well) and cultured for 4 days at 37°C, 5% CO2, in complete culture medium. 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 with 150 µL of supplemented lysis buffer #1 for 30 minutes 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.

2. 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 minutes 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 minutes 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.

3. 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 minutes 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.

4. 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 minutes 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
IRS2 total kit - 500 tests64IRTPEG
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IRS2 total kit - 10,000 tests64IRTPEH
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Companion products

DescriptionCat. noProduct insertMSDS
IRS2 total kit - control lysate64IRTTDA
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Literature