Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332

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Kit Includes Volume Solution Color
IRS-1 Ab Coated Microwells 96 tests
P-IRS-1 (S612) Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 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

H R

Reactivity Key:  H=Human  R=Rat
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1 when phosphorylated at Ser612. 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, a Phospho-IRS-1 (Ser612) Mouse Detection Antibody is added to detect phosphorylation of Ser612 on 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 the absorbance for this developed color is proportional to the quantity of IRS-1 phosphorylated at Ser612.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-IRS-1 (Ser612) Sandwich ELISA Kit #7332 detects endogenous levels of Phospho-IRS-1 when phosphorylated at Ser612. 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. 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). 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 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 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.

Sensitivity

Sensitivity

Figure 2. The relationship between 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.

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 the 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, suggesting 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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Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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