The relationship between protein concentration of lysates from untreated and insulin-treated CHO-IR/IRS-1 cells and the absorbance at 450 nm using PathScan® Phospho-Insulin Receptor β (Tyr1146) Sandwich ELISA Antibody Pair is shown. After starvation, CHO-IR/IRS-1 cells (85% confluence) were treated with 100 nM insulin for 2 min at 37°C and then lysed.
|7827S||1 Kit (Reagents for 4 x 96 well plates)||$ 469|
|Product Includes||Volume||Cap Color|
|Phospho-Insulin Receptor β (Tyr1146) Rabbit Capture Antibody (100X)||400 µl||Pink|
|Insulin Receptor β Detection Mouse mAb (100X)||400 µl||Blue|
|Anti-mouse IgG, HRP-linked Antibody (1000X)||40 µl||Yellow|
Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
posted January 2008
revised Sepetember 2013
Protocol Id: 20
CST's PathScan® Phospho-Insulin Receptor β (Tyr1146) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-Insulin Receptor β (Tyr1146) Sandwich ELISA Kit #7254. Capture and detection antibodies (100X stocks) and HRP-linked secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The phospho-insulin receptor β (Tyr1146) capture antibody is coated on a 96 well microplate in PBS overnight. After blocking, cell lysates are added followed by an insulin receptor β detection antibody and anti-mouse IgG, HRP-linked antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-insulin receptor β (Tyr1146) protein.
Antibodies in kit are custom formulations specific to kit.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair 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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