Figure 1: Treatment of CHO-IR/IRS-1 cells with insulin stimulates phosphorylation of insulin receptor at Tyr1146, detected by PathScan® Phospho-Insulin Receptor (Tyr1146) Sandwich ELISA Kit #7254, but the levels of total insulin receptor β shown by Western analysis remain unchanged. OD 450 readings are shown in the top figure, while the corresponding Western blots using Phospho-Insulin Receptor β (Tyr1146) Rabbit mAb #3021 (right panel) or Insulin Receptor β Antibody #3025 (left panel), are shown in the bottom figure. Human insulin receptor (IR) and rat IRS-1 are coexpressed in CHO-IR/IRS-1 cells.
Figure 2: The relationship between protein concentration of lysates from untreated and insulin-treated CHO-IR/IRS-1 cells and kit assay optical density readings is shown. After starvation, CHO-IR/IRS-1 cells (85% confluence) were treated with insulin (100 nM) for 2 min at 37°C, and then lysed.
|Product Includes||Volume||Solution Color|
|Phospho-Insulin Receptor (Tyr1146) Rabbit Antibody Coated Microwells||96 tests|
|Insulin Receptor ß Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Insulin Receptor β (Tyr1146) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfected phospho-insulin receptor (Tyr1146) protein. A Phospho-Insulin Receptor (Tyr1146) Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-insulin receptor (Tyr1146) proteins are captured by the coated antibody. Following extensive washing, Insulin Receptor β Mouse mAb is added to detect the captured phospho-insulin receptor (Tyr1146) protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-insulin receptor (Tyr1146) protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Insulin Receptor β (Tyr1146) Sandwich ELISA Kit #7254 detects transfected levels of phospho-insulin receptor β (Tyr1146) protein. As shown in Figure 1, using this ELISA Kit #7254, a significant induction of phospho-insulin receptor (Tyr1146) is detected in CHO-IR/IRS-1 cells treated with insulin. The levels of total insulin receptor β (phospho and nonphospho) shown by Western analysis remain unchanged. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Receptor autophosphorylation follows binding of the IGF-I and IGF-II ligands. Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).
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