Figure 1. Treatment of CHO (IR/4PS) cells with insulin stimulates tyrosine phosphorylation of IRS-2, detected by the PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA Kit #7860, but does not affect the level of total IRS-2 detected by Western analysis. CHO (IR/4PS) cells (80-90% confluent) were starved overnight and treated with 100 nM insulin for 10 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure. Kit specificity is demonstrated in the bottom figure by Western analysis of the ELISA microwell captured protein. Lysates were prepared from CHO (IR/4PS) cells and incubated in microwells coated with the IRS-2 capture antibody. The wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of CHO (IR/4PS) cell starting lysate (lanes 1, 2, 5 & 6) and the captured protein (lanes 3, 4, 7 & 8) was performed using IRS-2 Antibody #4502 (left panel) and Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 (right panel). The major protein detected in the captured material corresponds to IRS-2 (lanes 3, 4 & 8).
Figure 2. The relationship between the protein concentration of the lysate from untreated and insulin-treated CHO (IR/4PS) cells and the absorbance at 450 nm is shown.
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|Product Includes||Volume||Solution Color|
|IRS-2 Rabbit Antibody Coated Microwells||96 tests|
|Phospho Tyrosine 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|
The PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-2 when tyrosine phosphorylated. An IRS-2 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, IRS-2 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Tyrosine Mouse Detection Antibody is added to detect tyrosine phosphorylation of the captured IRS-2 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 the absorbance for this developed color is proportional to the quantity of IRS-2 phosphorylated on tyrosine.
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-IRS-2 (panTyr) Sandwich ELISA Kit #7860 detects IRS-2 when tyrosine phosphorylated. As shown in Figure 1, a significant induction of IRS-2 tyrosine phosphorylation can be detected in CHO (IR/4PS) cells following treatment with insulin using the Phospho-IRS-2 (panTyr) Sandwich ELISA Kit #7860. The level of total IRS-2 (phospho and nonphospho) remains unchanged as shown by Western analysis. This kit also detects tyrosine phosphorylated IRS-2 in insulin treated 3T3-L1 adipocytes (data not shown). 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 Substrate 2 (IRS-2) is one of the major substrates of the insulin receptor kinase (1). In vertebrates, IRS-2 functions as a scaffolding protein to coordinate separate branches of the Insulin/IGF-signaling cascades (2). IRS-2 is essential for normal nutrient homeostasis because it mediates both peripheral insulin action and the effect of IGF-1 on B-cell growth. Mice lacking IRS-2 fail to maintain sufficient compensatory insulin secretion and develop diabetes as young adults (3).
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