HTRF Human & Mouse Total IRS1 Detection Kit, 500 Assay Points

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 proteins 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 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 Tantis 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.

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.