Figure 1: Treatment of T-47D cells with Human Neuregulin-1 (hNRG-1) #5218 stimulates tyrosine phosphorylation of HER4/ErbB4 protein, as detected by PathScan® Phospho-HER4/ErbB4 (panTyr) Sandwich ELISA Kit #13125, but does not affect the level of total HER4/ErbB4 detected by PathScan® Total HER4/ErbB4 Sandwich ELISA Kit #13010. The absorbance readings at 450 nm are shown in the top figure while corresponding western blots using HER4/ErbB4 (111B2) Rabbit mAb #4795 (left panel) and Phospho-HER4/ErbB4 (Tyr984) Antibody #3790 (right panel) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and hNRG-1-treated T-47D cells and the absorbance at 450 nm as detected by the PathScan® Total HER4/ErbB4 Sandwich ELISA Kit is shown. Starved T-47D cells (85% confluence) were treated with Human Neuregulin-1 (hNRG-1) #5218 (100 ng/ml) for 2-5 min at 37°C and then lysed.
|Product Includes||Volume||Solution Color|
|HER4/ErbB4 Rabbit mAb Coated Microwells||96 tests|
|HER4/ErbB4 (L10A4) 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® Total HER4/ErbB4 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of HER4/ErbB4 protein. A HER4/ErbB4 rabbit mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-HER4/ErbB4 proteins are captured by the coated antibody. Following extensive washing, a HER4/ErbB4 mouse antibody is added to detect both the captured phospho- and nonphospho-HER4/ErbB4 proteins. 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 absorbance for the developed color is proportional to the quantity of HER4/ErbB4 protein.
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® Total HER4/ErbB4 Sandwich ELISA Kit #13010 detects endogenous levels of HER4/ErbB4 protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Research studies have implicated the HER/ErbB receptor tyrosine kinase family in normal development, cardiac function and cancer (1,2). HER4/ErbB4, like other family members, has four ectodomains, a single transmembrane domain and a cytoplasmic tail containing the active tyrosine kinase domain (3). By binding to neuregulins and/or EGF family ligands, ErbB4 forms either a homodimer or heterodimer with other ErbB family members, which results in receptor activation and signaling (3). ErbB4 is ubiquitously expressed with the highest expression occurring in brain and heart. The expression of ErbB4 in breast cancer, pediatric brain cancer and other types of carcinomas has been reported in research studies suggesting that ErbB4 expression is involved in both normal tissue development and carcinogenesis (3).
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PathScan is a trademark of Cell Signaling Technology, Inc.