Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total IRS-2 Sandwich ELISA Kit #7884

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Kit Includes Volume Solution Color
IRS-2 Ab Coated Microwells 96 tests
IRS-2 Detection Ab 11 ml Green
HRP-linked Streptavidin 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

M

Reactivity Key:  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Total IRS-2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total IRS-2 (also called 4PS). An IRS-2 rabbit antibody has been coated onto the microwells. After incubation with cell lysates, IRS-2 (both phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a biotinylated IRS-2 rabbit antibody is added to detect the captured IRS-2. HRP-linked streptavidin 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.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total IRS-2 Sandwich ELISA Kit #7884 detects endogenous levels of IRS-2. As shown in Figure 1, using the PathScan® Total IRS-2 Sandwich ELISA Kit #7884, a high level of IRS-2 is detected in transfected CHO cells. The total levels of IRS-2 (phospho and nonphospho) remain unchanged after insulin treatment as shown by western analysis. This kit also detects endogenous levels of IRS-2 expressed by 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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of CHO (IR/4PS) cells with insulin stimulates phosphorylation of IRS-2 at tyrosine residues as detected by PathScan® Phospho-IRS-2 (panTyr) Sandwich ELISA #7861, but does not affect the level of total IRS-2 protein detected by PathScan® Total IRS-2 Sandwich ELISA #7884. CHO cells stably transfected with the insulin receptor and IRS-2 (CHO (IR/4PS) cells) were starved overnight and treated with 100 nM insulin for 2 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots are shown in the bottom figure. In order to detect tyrosine phosphorylated IRS-2, total IRS-2 was immunoprecipitated using microwells coated with an IRS-2-specific antibody. The wells were washed and the protein solubilized with SDS loading buffer. The captured protein was then separated by gel electrophoresis and probed with either IRS-2 Antibody #4502 (left panel) or Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 (right panel).

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the lysate from untransfected CHO cells or CHO cells stably transfected with the insulin receptor + IRS-2 and the absorbance at 450 nm is shown.

Background

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).

  1. Sun, X.J. et al. (1991) Nature 352, 73-77.
  2. White, M.F. (2002) Am. J. Physiol. Endocrinol. Metab. 283, E413-E422.
  3. Withers, D.J. et al. (1998) Nature 391, 900-904.

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