Figure 1. Constitutive phosphorylation of HER3/ErbB3 in Calu-3 cells lysed in the presence of phosphatase inhibitors* (phospho lysate) is detected by PathScan® Phospho-HER3/ErbB3 (panTyr) Sandwich ELISA Kit #7890 (top, right). In contrast, a low level of phospho-HER3/ErbB3 protein is detected in Calu-3 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysate). Similar levels of total HER3/ErbB3 protein from both nonphospho or phospho lysates are detected by PathScan® Total HER3/ErbB3 Sandwich ELISA Kit #7888 (top, left). Absorbance at 450 nm is shown in the top figure while corresponding western blots using Phospho-HER3/ErbB3 (Tyr1289) Rabbit mAb #4791 (right) or a total HER3/ErbB3 Antibody #4754 (left) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Figure 2. The relationship between protein concentration of lysates from untreated and NRG-treated T-47D cells and kit assay optical density readings. After starvation, T-47D cells (85% confluence) were treated with Human Neuregulin-1 (hNRG-1) #5218 (100 ng/ml) for 5 minutes at 37°C and then lysed.
|Product Includes||Volume||Solution Color|
|HER3/ErbB3 Rabbit mAb Coated Microwells||96 tests|
|Phospho Tyrosine Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-HER3/ErbB3 (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated HER3/ErbB3 protein. A HER3/ErbB3 rabbit mAb has been coated on the microwells. After incubation with cell lysates, HER3/ErbB3 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 HER3/ErbB3 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 HER3/ErbB3 protein phosphorylated on tyrosine.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-HER3/ErbB3 (panTyr) Sandwich ELISA Kit #7890 detects endogenous levels of HER3/ErbB3 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.
HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but it lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Upon ligand binding, heterodimers form between ErbB3 and other ErbB proteins, and ErbB3 is phosphorylated on tyrosine residues by the activated ErbB kinase (1,2). There are at least 9 potential tyrosine phosphorylation sites in the carboxy-terminal tail of ErbB3. These sites serve as consensus binding sites for signal transducing proteins, including Src family members, Grb2, and the p85 subunit of PI3 kinase, which mediate ErbB downstream signaling (3). Both Tyr1222 and Tyr1289 of ErbB3 reside within a YXXM motif and participate in signaling to PI3K (4).
Investigators have found that ErbB3 is highly expressed in many cancer cells (5) and activation of the ErbB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas (6). Research studies have demonstrated that in tumor development, ErbB3 may function as an oncogenic unit together with other ErbB members (e.g. ErbB2 requires ErbB3 to drive breast tumor cell proliferation) (7). Thus, investigators view inhibiting interaction between ErbB3 and ErbB tyrosine kinases as a novel strategy for anti-tumor therapy.
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PathScan is a trademark of Cell Signaling Technology, Inc.