Figure 1. Treatment of MCF-7 cells with IGF-I stimulates phosphorylation of IGF-I receptor at Tyr1131, detected by PathScan® Phospho-IGF-I Receptor β (Tyr1131) Sandwich ELISA Kit #7302, but does not affect the level of total IGF-I Receptor β protein detected by Western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using Phospho-IGF-I Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) Antibody #3021 (right panel) or IGF-I Receptor β Antibody #3027 (left panel), are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of untreated and IGF-I-treated MCF-7 cell lysates and the absorbance at 450 nm is shown. Cells were serum starved overnight and then treated with 100 nm IGF-I for 5 min. at 37°C.
|Product Includes||Volume||Solution Color|
|Phospho-IGF-I Receptor beta (Tyr1131) Rabbit Antibody Coated Microwells||96 tests|
|IGF-I Receptor 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-IGF-I Receptor beta (Tyr1131) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IGF-I receptor beta protein when phosphorylated at Tyr1131. A Phospho-IGF-I Receptor beta (Tyr1131) Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-IGF-I Receptor beta is captured by the coated antibody. Following extensive washing, an IGF-I Receptor Mouse Antibody is added to detect the captured phospho-IGF-I receptor 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 absorbance for this developed color is proportional to the quantity of IGF-I receptor protein phosphorylated at Tyr1131.
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-IGF-I Receptor β (Tyr1131) Sandwich ELISA Kit #7302 detects endogenous levels of IGF-I Receptor β protein when phosphorylated at Tyr1131. A significant induction of phosphorylation of IGF-I receptor at Tyr1131 is detected in IGF-I- treated MCF-7 cells using PathScan® Phospho-IGF-I Receptor β (Tyr 1131) Sandwich ELISA Kit #7302 (Figure 1). The levels of total IGF-I receptor β (phospho and nonphospho) shown by Western analysis remain unchanged (Figure 1). 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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