Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total IRS-1 Sandwich ELISA Kit #7328

Kit Includes Volume Solution Color
IRS-1 Rabbit Ab Coated Microwells 96 tests
IRS-1 Mouse Detection Ab 11 milliliters Green
Anti-Mouse IgG HRP-Linked Ab 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters 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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H R

Reactivity Key:  H=Human  R=Rat

Description

The PathScan® Total IRS-1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1. An IRS-1 Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, IRS-1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, IRS-1 Mouse Detection Antibody* is added to detect the captured IRS-1 protein. Anti-mouse IgG, HRP-linked Antibody #7076* 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 total IRS-1.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total IRS-1 Sandwich ELISA Kit #7328 detects endogenous levels of IRS-1. As shown in Figure 1, a significant induction of IRS-1 phosphorylation at Ser612 can be detected in hSkMC and CHO (IR/IRS-1) cells following treatment with insulin using the Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332. However, the level of total IRS-1 (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total IRS-1 Sandwich ELISA Kit #7328 (Figure 1). In Figure 3, Western analysis of protein captured in microwells coated with the IRS-1 antibody shows a major band corresponding to the IRS-1 protein.

Sandwich ELISA

Sandwich ELISA

Figure 1. Treatment of hSkMC or CHO (IR/IRS-1) cells with insulin stimulates phosphorylation of IRS-1 at Ser612, detected by the PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332, but does not affect the level of total IRS-1 protein detected by PathScan® Total IRS-1 Sandwich ELISA Kit #7328. hSkMC and CHO (IR/IRS-1) cells (80-90% confluent) were starved overnight and treated with 100 nM insulin for 7 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using IRS-1 (L3D12) Mouse mAb #3194 (panels A & B) or Phospho-IRS-1 (Ser612) (L7B8) Mouse mAb #3193 (panels C & D), are shown in the bottom figure.

Sandwich ELISA

Sandwich ELISA

Figure 2. The relationship between the lysate protein concentration from untreated and insulin-treated CHO (IR/IRS-1) cells (A) or hSkMC cells (B) and the absorbance at 450 nm is shown.

Sandwich ELISA

Sandwich ELISA

Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from CHO (IR/IRS-1) cells and incubated in microwells coated with the IRS-1 capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of CHO (IR/IRS-1) cell starting lysate (lanes 1 & 2) and the captured protein (lanes 3 & 4) was performed using IRS-1 (L3D12) Mouse mAb #3194. The major band detected in the captured material corresponds to IRS-1 (lanes 3 & 4).


Background

Insulin receptor substrate 1 (IRS-1) is one of the major substrates of the insulin receptor kinase (1). IRS-1 contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2 domain containing proteins that mediate the metabolic and growth promoting functions of insulin (2-4). IRS-1 also contains over 30 potential serine/threonine phosphorylation sites. Ser307 of IRS-1 is phosphorylated by JNK (5) and IKK (6) while Ser789 is phosphorylated by SIK-2, a member of AMPK family (7). The PKC and mTOR pathways mediate phosphorylation of IRS-1 at Ser612 and Ser636/639, respectively (8,9). Phosphorylation of IRS-1 at Ser1101 is mediated by PKCθ and results in an inhibition of insulin signaling in the cell, which suggests a potential mechanism for insulin resistance in some models of obesity (10).

  1. Sun, X.J. et al. (1991) Nature 352, 73-77.
  2. Sun, X.J. et al. (1992) J. Biol. Chem. 267, 22662-22672.
  3. Myers Jr., M.G. et al. (1993) Endocrinology 132, 1421-1430.
  4. Wang, L.M. et al. (1993) Science 261, 1591-1594.
  5. Rui, L. et al. (1997) J. Clin. Invest. 107, 181-189.
  6. Gao, Z. et al. (2002) J. Biol. Chem. 277, 48115-48121.
  7. Horike, N. et al. (2003) J. Biol. Chem. 278, 18440-18447.
  8. Ozes, O.N. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4640-4645.
  9. De Fea, K. and Ruth, R.A. (1997) Biochemistry 36, 12939-12947.
  10. Li, Y. et al. (2004) J. Biol. Chem. 279, 45304-45307.

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