The phospho-IRS1 (Ser307) assay enables the cell-based detection of Ser307 phosphorylation on IRS1.
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  • High sensitivity
  • Faster and more convenient than ELISA
The phospho-IRS1 (Ser307) assay enables the cell-based detection of Ser307 phosphorylation on IRS1.
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Overview

This HTRF cell-based assay conveniently and accurately quantifies phosphorylated IRS1 at (Ser307 mouse/Ser312 human). IRS1 is a relevant marker for insulin resistance, tumor development, and metastatic progression. It is associated with type 2 diabetes, obesity, and cancer.

Total-IRS1 assay principle

The total IRS1 assay quantifies the expression level of IRS1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The total IRS1 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 IRS1 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-IRS1 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 IRS1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-IRS1 1-plate assay protocol

Detection of total IRS1 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.

Total IRS1 assay validation on C2C12 mouse myotubes

C2C12 cells were cultured until 100% confluency was reached. They were then cultured for 7 days in 2% horse serum media to induce the differentiation. After a starvation step in serum-free medium, cells were treated with a cocktail of pro-inflammatory cytokines and/or Insulin. 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.

Total IRS1 assay validation on 3T3-L1 mouse adipocytes

3T3-L1 cells were cultured in medium with 10% newborn calf serum. They were then cultured for 7 days in medium containing 10% FCS, insulin, dexamethasone, IBMX and µM thiazolidinedione to induce 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 for 30 min. After cell supernatant removal, cells were lysed and 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, UMR INSERM U1065/UNS, C3M, Nice, France).

Total IRS1 assay 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.

Phospho-IRS1 assay 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.

IRS1/PI3K/AKT signaling pathway

Modulation of the IRS1/PI3K/AKT signaling pathway

Inhibition of the pathway. While tyrosine-phosphorylated IRS-1 positively regulates insulin signaling, serine phosphorylation generally serves as negative feedback to down-regulate IRS-1 function. Increased serine phosphorylation of IRS-1 represents a hallmark of insulin resistance in adipose tissue and skeletal muscles. Binding of Insulin or IGF-1 to the extracellular a-subunits of the IR or IGF-1R triggers autophosphorylation of the transmembrane ß-subunits of the receptor, thus activating it. 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. IRS1 downregulation is a relevant diabetic marker while overexpression is associated with metastatic growth and cancers.
Activation of the pathway. Binding of Insulin or IGF-1 to the extracellular a-subunits of the IR or IGF-1R (or to hybrids of both receptors) triggers autophosphorylation of the transmembrane ß-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. The PI3K pathway is then activated with downstream phosphorylations of AKT, followed by 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, while upregulation of the IGF-1/IRS1 pathway upon IRS1 overexpression, is a readout for tumor development and metastatic progression.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert IRS1 Kit / 64NRSPEG

64NRSPEG - Product Insert

Product Insert IRS1 Kit / 64NRSPEH

64NRSPEH - Product Insert

Product Insert IRS1 Kit / 64IRSPEG-64IRSPEH

64IRSPEG-64IRSPEH - Product Insert

Product Insert IRS1 Kit / 64NRSPEG-64NRSPEH

64NRSPEG-64NRSPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

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