Figure 1. Detection of EpCAM protein MCF7, HT-29, HeLa cells is examined by using the PathScan® Total EpCAM Sandwich ELISA Kit. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blot using EpCAM (D1B3) Rabbit mAb #2626 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from HT-29 cells and the absorbance at 450 nm as detected by the PathScan® Total EpCAM Sandwich ELISA Kit is shown. Unstarved HT-29 cells (85% confluence) were harvested and then lysed.
|Product Includes||Volume||Solution Color|
|EpCAM Rabbit mAb Coated Microwells||96 tests|
|EpCAM 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 EpCAM Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of EpCAM protein. An EpCAM rabbit mAb has been coated onto the microwells. After incubation with cell lysates, the EpCAM proteins are captured by the coated antibody. Following extensive washing, an EpCAM mouse detection mAb is added to detect captured EpCAM proteins. Anti-mouse, 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 EpCAM protein.
Antibodies in the kit are custom formulations specific to the 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 EpCAM Sandwich ELISA Kit detects endogenous levels of EpCAM 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.
Epithelial cell adhesion and activating molecule (EpCAM/CD326) is a transmembrane glycoprotein that mediates Ca2+-independent, homophilic adhesions on the basolateral surface of most epithelial cells. EpCAM is not expressed in adult squamous epithelium, but it is highly expressed in adeno and squamous cell carcinomas (1). Research studies identified EpCAM as one of the first tumor-associated antigens, and it has long been a marker of epithelial and tumor tissue. Investigators have shown that EpCAM is highly expressed in cancer cells (reviewed in 2,3).
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