Product Pathways - PathScan ELISA
PathScan® Phospho-HER2/ErbB2 (panTyr) Sandwich ELISA Kit #7968
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|Kit Includes||Volume||Solution Color|
|HER2/ErbB2 Rabbit mAb Coated Microwells||96 tests|
|Phospho-Tyrosine Mouse Detection mAb||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|
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 enclosed in parentheses are predicted to react based on 100% sequence homology.
The PathScan® Phospho-HER2/ErbB2 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated HER2/ErbB2 protein. A HER2/ErbB2 rabbit mAb has been coated on the microwells. After incubation with cell lysates, HER2/ErbB2 protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection antibody is added to detect captured tyrosine-phosphorylated HER2/ErbB2 protein. Anti-mouse IgG, HRP-linked antibody 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 HER2/ErbB2 protein phosphorylated on tyrosine.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Phospho-HER2/ErbB2 (panTyr) Sandwich ELISA Kit #7968 detects endogenous levels of HER2/ErbB2 protein only when phosphorylated at Tyr residues (see Figure 1). The kit sensitivity is shown in Figure 2. This kit does not cross-react with other proteins of ErbB family (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
Figure 1: Constitutive phosphorylation of HER2/ErbB2 in Calu-3 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-HER2/ErbB2 (panTyr) Sandwich ELISA Kit #7968 (top, right). In contrast, a low level of phospho-HER2/ErbB2 protein is detected in Calu-3 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total HER2/ErbB2 protein from both nonphospho or phospho lysates are detected by PathScan® Total HER2/ErbB2 Sandwich ELISA Kit #7310 (top, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Phospho-HER2/ErbB2 (Tyr1221/1222) Rabbit mAb #2243 (right) or a total HER2/ErbB2 Antibody #2242 (left) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Figure 2: The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm is shown. Unstarved Calu-3 cells were cultured (85% confluence) and lysed with or without the addition of phosphatase inhibitor to the lysis buffer (phospho or nonphospho lysate).
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).
- Muthuswamy, S.K. et al. (1999) Mol Cell Biol 19, 6845-57.
- Qian, X. et al. (1994) Proc Natl Acad Sci USA 91, 1500-4.
- Dittadi, R. and Gion, M. (2000) J Natl Cancer Inst 92, 1443-4.
- Klapper, L.N. et al. (2000) Cancer Res 60, 3384-8.
- Kwon, Y.K. et al. (1997) J Neurosci 17, 8293-9.
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- 7310 PathScan® Total HER2/ErbB2 Sandwich ELISA Kit
- 7148 PathScan® Phospho-HER2/ErbB2 (Tyr1221/1222) Sandwich ELISA Kit
- 2244 Phospho-HER2/ErbB2 (Tyr1248)/EGFR (Tyr1173) Antibody
- 2241 Phospho-HER2/ErbB2 (Tyr877) Antibody
- 6942 Phospho-HER2/ErbB2 (Tyr1196) (D66B7) Rabbit mAb
- 2243 Phospho-HER2/ErbB2 (Tyr1221/1222) (6B12) Rabbit mAb
- 2249 Phospho-HER2/ErbB2 (Tyr1221/1222) Antibody
- 2247 Phospho-HER2/ErbB2 (Tyr1248) Antibody
- 4290 HER2/ErbB2 (D8F12) XP® Rabbit mAb
- 2242 HER2/ErbB2 Antibody
- 9803 Cell Lysis Buffer (10X)
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9998 BSA
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.