Phospho-IRS1 (Ser307 mouse / Ser312 human) & Total IRS1 Cellular Assay Kits

Phospho- and total IRS1 assays for monitoring Insulin resistance

Cisbio's cell-based homogeneous HTRF® phospho- and total IRS1 assays enable quantitative detection of the modulation of IRS1 phosphorylated on Serine 307 in mouse cells and on Serine 312 in human cells.

IRS1, relevant marker for insulin resistance, tumor development and metastatic progression, is associated with type 2 diabetes, obesity and cancer. Using a streamlined protocol without any washing steps and amenable to low-volume formats, phospho- and total IRS1 assays can be used from basic research up to preclinical drug discovery phases. These kits contains all the reagents you need, and offer increased throughput compared to ELISA or Western Blot.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total IRS1 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-IRS1 antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with IRS1, and the proximity of donor and acceptor will then lead 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 assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-IRS1 can be quantitatively detected using the HTRF phospho-IRS1 (Ser307 mouse / Ser312 human) and total IRS1 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

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 to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Product performance

1. Validation on C2C12 mouse myotubes

C2C12 mouse myoblasts were plated in  6-well plates and cultured for 2 days in complete culture medium with 10% FCS until 100% confluency was reached. Cells were then cultured for 7 days in medium containing 2% horse serum to induce the differentiation from myoblasts to myotubes. After a starvation step for 5h in serum-free medium, cells were treated with a cocktail of pro-inflammatory cytokines (50 ng/mL TNF-α + 50 ng/mL IL-1β for 1h) and/or Insulin (100 nM for 10 min or 45 min). After cell supernatant removal, cells were lysed with 1.5 mL of supplemented lysis buffer #1 for 30 min at RT under gentle agitation. 16 µL of lysate were then transferred into a 384-well low volume white microplate for HTRF detection using the phospho- and total IRS1 kit reagents.

2. Validation on 3T3-L1 mouse adipocytes

3T3-L1 mouse fibroblasts were plated in 12-well plates and cultured at 37°C, 7% CO2 in complete culture medium with 10% newborn calf serum. Cells were then cultured for 7 days in medium containing 10% FCS, 5 µg/mL insulin, 0.25 µM dexamethasone, 0.5 mM IBMX and 10 µM thiazolidinedione to induce the differentiation from fibroblasts to adipocytes. After an overnight starvation step in serum-free medium, the cells were treated with 100 nM insulin for 45 min or with a cocktail of pro-inflammatory cytokines (50 ng/mL TNF-α + 50 ng/mL IL-1β) for 30 min. After cell supernatant removal, cells were lysed with 600 µL of supplemented Lysis Buffer #1 for 30 min at RT under gentle agitation. 16 µL of lysate were then transferred into a 384-well low volume white microplate for HTRF detection using the phospho- and total IRS1 kit reagents.

Samples were kindly provided by JF Tanti’s research team “Cellular and Molecular Physiopathology of Obesity and Diabetes”, UMR INSERM U1065/UNS, C3M, Nice.

3. Validation on MCF-7 human breast cancer cells

50,000 human breast cancer MCF-7 cells were plated in a 96-well plate in complete culture medium, and incubated for 24h at 37°C, 5% CO2. Increasing concentrations of Insulin were added and incubated for 45 min at 37°C.

After cell culture medium removal, cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were then transferred into a 384-well sv white microplate for detection of phospho-IRS1 Ser 312 and total IRS1, using 4 µL of the HTRF detection reagents.

4. HTRF assay compared to Western Blot using phospho IRS1 cellular assay on 3T3-L1 mouse adipocytes

3T3-L1 mouse fibroblasts were plated in 12-well plates and cultured at 37°C, 7% CO2 in complete culture medium with 10% newborn calf serum. Cells were then cultured for 7 days in medium containing 10% FCS, 5 µg/mL insulin, 0.25 µM dexamethasone, 0.5 mM IBMX and 10 µM thiazolidinedione to induce the differentiation from fibroblasts to adipocytes.

After an overnight starvation step in serum-free medium, the cells were treated with insulin for 45 min or with a cocktail of pro-inflammatory cytokines (10 ng/mL TNF-α + 10 ng/mL IL-1β) for 30 min. After cell supernatant removal, cells were lysed with 600 µL of supplemented lysis buffer #1 for 30 min at RT under gentle agitation.

After a 10 min centrifugation step, 16 µL of lysate were transferred in triplicate into a 384-well plate for HTRF detection or into a gel for WB detection. Data presented on the graph correspond to the mean of each triplicate.

The HTRF assay provides higher pharmacological assay windows than Western Blot.

Simplified pathway

IRS1/PI3K/AKT signaling pathway

Inhibition of the IRS1/PI3K/AKT signaling pathway

While tyrosine-phosphorylated IRS-1 positively regulates insulin signaling, serine phosphorylation generally serves as a negative feedback to down-regulate IRS-1 function by promoting dissociation of IRS-1 from the receptor, blockage of specific tyrosine phosphorylation sites, and degradation of IRS-1.

Diabetogenic factors such as pro-inflammatory cytokines, free fatty acids, and hyperinsulinemia, increase the serine phosphorylation of IRS-1, which therefore represents a hallmark of insulin resistance in adipose tissue and skeletal muscles. One of the more notable phospho-sites in humans is Ser312 (= Ser307 in mouse). It constitutes a key regulatory site that disrupts the IRS1/IR interaction and inhibits insulin-mediated activation of the PI3K pathway. This residue is directly phosphorylated by activated JNK and IKK stress proteins. Phosphorylation on Ser312 is also mediated by the mTOR pathway and acts as a negative feedback loop during prolonged insulin exposure.

Activation of the IRS1/PI3K/AKT signaling pathway

Binding of Insulin or IGF-1 to the extracellular α-subunits of the IR or IGF-1R (or to hybrids of both receptors) triggers autophosphorylation of the transmembrane β-subunits of the receptor.

IRS-1 is then recruited by the activated tyrosine kinase receptor and phosphorylated on multiple tyrosine residues, creating a docking site for PI3K at the membrane.

PI3K then activates PDK1 which in turn phosphorylates AKT on Thr308.

AKT directly phosphorylates its substrates AS160, GSK3 and FoxO1, leading respectively to GLUT4 translocation/glucose uptake, glycogen synthesis and inhibition of gluconeogenesis. Thus, the IRS1/AKT pathway activation plays a critical role in maintaining glucose homeostasis. AKT also activates mTOR that mediates cell growth and survival, as well as protein synthesis.

Related disorders

IRS1 phosphorylation and degradation, which lead to the inactivation of the Insulin/IRS1 pathway, make it a relevant marker for insulin resistance in skeletal muscles cells and adipocytes.

IRS1 overexpression, which leads to an upregulation of the IGF-1/IRS1 pathway, makes it a relevant marker for tumor development and metastatic progression.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
IRS1 phospho-Ser312 kit - 500 tests64IRSPEG
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IRS1 phospho-Ser312 kit - 10,000 tests64IRSPEH
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IRS1 phospho-Ser312 kit - 50,000 tests64IRSPEY
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IRS1 total kit - 500 tests64NRSPEG
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IRS1 total kit - 10,000 tests64NRSPEH
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IRS1 total kit - 50,000 tests64NRSPEY
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Companion products

DescriptionCat. noProduct insertMSDS
IRS1 phospho-Ser312 kit control lysate64IRSTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
IRS1 total kit control lysate64NRSTDA
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