|7148||1 Kit ( 96 assays )|
|Kit Includes||Volume||Solution Color|
|HER2/ErbB2 mAb coatedmicrowells||96 tests|
|P-HER2/ErbB2 (Y1221/ 1222) Detection Ab||11 milliliters||Green|
|Anti-rabbit IgG HRP-Linked Ab||11 milliliters||Red|
|TMB Substrate||11 milliliters||Colorless|
|STOP Solution||11 milliliters||Colorless|
|20X Wash Buffer||25 milliliters||Colorless|
|Sample Diluent||25 milliliters||Blue|
|Sealing Tape||2 sheets|
|Cell Lysis Buffer (10X) # 9803||15 milliliters|
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
Reactivity Key: H=Human
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 #2170* 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 #2243* 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.
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 (37°C 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.
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.
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 U S A 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.
7148: Sandwich ELISA
* 2243 Phospho-HER2/ErbB2 (Tyr1221/1222) (6B12) Rabbit mAb
* 7074 Anti-rabbit IgG, HRP-linked Antibody
* 2242 HER2/ErbB2 Antibody
Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.