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phospho-IRS1 (Ser312) STAR ELISA kit
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PRODUCT FAMILY INFORMATION
STAR ELISA Kits
Upstate STAR (Signal Transduction Assay Reaction) ELISA kits are a fast, sensitive method to detect the relative amounts of total and activated targets through the use of phospho-specific antibodies.
Upstate STAR (Signal Transduction Assay Reaction) ELISA kits are a fast, sensitive method to detect the relative amounts of total and activated targets through the use of phospho-specific antibodies.
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Description:
phospho-IRS1 (Ser307 mouse/ Ser312 Human) STAR ELISA Kit
Trade Name:
STAR ELISA
Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
I. TEST PRINCIPLE
The UPSTATE® colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The IRS1 plate is coated with a specific mouse monoclonal capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing IRS1 antigen to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-IRS1 (Ser312) antibody to detect the captured IRS1 on the plate well that is phosphorylated on Ser312. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard curve.
II. IRS1 BACKGROUND
IRS (Insulin Receptor Substrate) proteins are the effectors of both Insulin and IGF-initiated signaling. Insulin, in addition to working very similarly to a growth factor in stimulating receptor tyrosine kinases, is essential for maintaining glucose homeostasis and regulating carbohydrate, lipid, and protein metabolism. The signaling events of insulin are mediated via a vast and highly integrated network of molecules that controls several processes. There are 4 IRS proteins, 1-4. It is believed that these proteins serve complementary functions in different tissues. The two prominent players are IRS1 and IRS2 with IRS1 playing important roles in skeletal muscle and IRS2 in the liver. The IRS proteins link the insulin receptor with many signaling pathways including the PI3 Kinase/Akt/mTOR and the MAP Kinase pathways. The PI3K/Akt/mTOR pathway is involved with many metabolic actions of insulin signaling. MAPK is involved in the regulation of some genes and works with PI3 Kinase to control cell growth and differentiation. In addition to the insulin receptor tyrosine kinase activity in type II diabetes patients and mice, studies have shown a greater defect in components of the insulin signaling pathway including Insulin Receptor Substrate (IRS) phosphorylation or the activation of Phosphoinositide 3- Kinase (PI3 Kinase). Insulin stimulates the signaling cascade by binding to the insulin receptor through its extracellularα subunits. This stimulates the tyrosine kinase activity of the β subunits of the receptor. The receptor then autophosphorylates itself and phosphorylates the IRS (Insulin Receptor Substrate) proteins that are very important modulators of insulin signaling. IRS binds to the phosphorylated tyrosines of the β subunit of the insulin receptor. They share PH and PTB domains near their N-termini, and m |