Figure 1: Treatment of CHO-IR/IRS-1 cells with insulin stimulates tyrosine phosphorylation of Insulin Receptor β which is detected by PathScan® Phospho-Insulin Receptor β (panTyr) Sandwich ELISA Kit #7082 (top, right). Similar levels of Insulin Receptor β protein from both nonphospho or phospho lysates are detected by PathScan® Total Insulin Receptor β Sandwich ELISA Kit #7069 (top, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Phospho-IGF-I Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) (19H7) Rabbit mAb #3024 (right panel) or Insulin Receptor β (4B8) Rabbit mAb #3025 (left panel) are shown in the bottom figure. CHO-IR/IRS-1 cells stably overexpress the tranfected human insulin receptor and rat IRS-1.
Figure 2: The relationship between protein concentration of lysates from untreated and insulin-treated CHO-IR/IRS-1 cells and the absorbance at 450 nm 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|
|Insulin Receptor β Mouse mAb Coated Microwells||96 tests|
|Insulin Receptor beta (D11G10) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit 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|
PathScan® Total Insulin Receptor β Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfected levels of Insulin Receptor β protein. An Insulin Receptor β Mouse mAb has been coated on the microwells. After incubation with cell lysates, Insulin Receptor β protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, an Insulin Receptor β Rabbit mAb is added to detect captured Insulin Receptor β protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Insulin Receptor β 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
PathScan® Total Insulin Receptor β Sandwich ELISA Kit #7069 detects transfected levels of human Insulin Receptor β protein, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Insulin receptor (INSR) is a membrane receptor tyrosine kinase. The receptor molecule consists of a disulfide linked heterodimer. The α subunit is a 135 kDa extracellular fragment, and the β subunit is a 95 kDa fragment containing an extracellular domain, a single transmembrane domain, and an intracellular tyrosine kinase domain (1). Insulin ligand binding to this receptor results in receptor autophosphorylation and tyrosine kinase activation. INSR catalyzes the tyrosine phosphorylation of molecules such as IRS, Gab1, Shc, and Cbl, which further activate the downstream MAPK, PI3K, and TC10 pathways. This eventually leads to increases in glucose uptake and metabolism as well as cell growth (2,3). INSR has peptide substrate specificity similar to other receptor tyrosine kinase members, preferring acidic residues at the -1 to -4 positions and large hydrophobic amino acids at positions +1 and +3 (4).
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