Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA Kit #7148

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Kit Includes Volume Solution Color
HER2/ErbB2 mAb coatedmicrowells 96 tests
P-HER2/ErbB2 (Y1221/ 1222) Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Sealing Tape 2 sheets
Cell Lysis Buffer (10X) #9803 15 ml

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

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

Description

CST's PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-HER2/ErbB2 (Tyr1221/1222) protein. A HER2/ErbB2 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-HER2/Erb2 Receptor proteins are captured by the coated antibody. Following extensive washing, Phospho-HER2/ErbB2 (Tyr1221/1222) Antibody is added to detect the captured phospho-HER2/ErbB2 protein. Anti-rabbit IgG, HRP-linked Antibody #7074 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-HER2/ErbB2 (Tyr1221/1222) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA Kit detects endogenous levels of phospho-HER2/ErbB2 (Tyr1221/1222) protein. As shown in Figure 1, using PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA Kit, a high level of phospho-HER2/ErbB2 (Tyr1221/1222) protein is detected in Calu-3 cells, and the levels are significantly reduced in Calu-3 cells treated with λ protein phosphastase. 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

Figure1: Phosphorylation of HER2/ErbB2 at Tyr1221/1222 detected by PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA kit #7148. HER2/ErbB2 receptor kinases in Calu-3 cells are known to be constitutively activated. Treatment of Calu-3 cell lysates with λ protein phosphatase (37ºC for 60 minutes) abolished the phosphorylation of HER2/ErbB2 in the lysates shown in both sandwich ELISA and Western analysis. OD 450 readings are shown in the top figure, while the corresponding Western blots, using Phospho-HER2/ErbB2 (Tyr1221/1222) Antibody #2243 (right panel) or HER2/ErbB2 Antibody #2242 (left panel), is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of untreated and LPP-treated Calu-3 cell lysates (see the legend of Figure 1) and kit assay optical density readings.

Background

The ErbB2 (HER2) proto-oncogene encodes a 185 kDa transmembrane, receptor-like glycoprotein with intrinsic tyrosine kinase activity (1). While ErbB2 lacks an identified ligand, ErbB2 kinase activity can be activated in the absence of a ligand when overexpressed and through heteromeric associations with other ErbB family members (2). Amplification of the ErbB2 gene and overexpression of its product are detected in almost 40% of human breast cancers (3). Binding of the c-Cbl ubiquitin ligase to ErbB2 at Tyr1112 leads to ErbB2 poly-ubiquitination and enhances degradation of this kinase (4). ErbB2 is a key therapeutic target in the treatment of breast cancer and other carcinomas and targeting the regulation of ErbB2 degradation by the c-Cbl-regulated proteolytic pathway is one potential therapeutic strategy. Phosphorylation of the kinase domain residue Tyr877 of ErbB2 (homologous to Tyr416 of pp60c-Src) may be involved in regulating ErbB2 biological activity. The major autophosphorylation sites in ErbB2 are Tyr1248 and Tyr1221/1222; phosphorylation of these sites couples ErbB2 to the Ras-Raf-MAP kinase signal transduction pathway (1,5).

  1. Muthuswamy, S.K. et al. (1999) Mol Cell Biol 19, 6845-57.
  2. Qian, X. et al. (1994) Proc Natl Acad Sci USA 91, 1500-4.
  3. Dittadi, R. and Gion, M. (2000) J Natl Cancer Inst 92, 1443-4.
  4. Klapper, L.N. et al. (2000) Cancer Res 60, 3384-8.
  5. Kwon, Y.K. et al. (1997) J Neurosci 17, 8293-9.

Application References

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Protocols

Companion Products

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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