Figure 2: The relationship between protein concentration of lysates from untreated and EGF-treated A431 cells and kit assay optical density readings. After starvation, A431 cells (85% confluence) were treated with EGF (100 ng/ml) for 5 min at 37°C, and then lysed.
Figure 1: Treatment of A431 cells with EGF stimulates phosphorylation of EGF Receptor at Tyr1068, detected by PathScan® Phospho-EGF Receptor (Tyr1068) Sandwich ELISA kit #7240, but does not affect the level of total EGF Receptor detected by PathScan® Total EGF Receptor Sandwich ELISA kit #7250. OD 450nm readings are shown in the top figure, while the corresponding western blot using Phospho-EGF Receptor (Tyr1068) Antibody #2234 (right panel) or EGF Receptor Antibody #2232 (left panel), is shown in the bottom figure.
Figure 3: Kit specificity demonstrated by western blot analysis of the ELISA-well captured protein. Lysates prepared from human A431 cells were incubated in wells coated with an EGF Receptor capture antibody. Wells were washed and captured protein was solubilized in SDS gel loading buffer. A431 lysate (lane 1) and captured protein (lane 2) were analyzed by western blot using an EGF Receptor detection antibody. A single band corresponding to the EGF Receptor is detected in the captured material (lane 2).
|Product Includes||Volume||Solution Color|
|EGF Receptor Mouse mAb Coated Microwells||96 tests|
|EGF Receptor Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit 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|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Total EGF Receptor Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total EGF Receptor protein. An EGF Receptor Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-EGF Receptor proteins are captured by the coated antibody. Following extensive washing, EGF Receptor Rabbit Antibody is added to detect both the captured phospho- and nonphospho-EGF Receptor protein. Anti-rabbit IgG, HRP-linked Antibody 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 total EGF Receptor 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
CST's PathScan® Total EGF Receptor Sandwich ELISA Kit #7250 detects endogenous levels of EGF Receptor Protein. As shown in Figure 1, using PathScan® Phospho-EGF Receptor (Tyr1068) ELISA Kit #7240, a significant induction of Phospho-EGF Receptor (Tyr1068) is detected in A-431 cells treated with EGF. The levels of total EGF Receptor (phospho and non-phospho) detected by PathScan® Total EGF Receptor Sandwich ELISA Kit #7250 remain unchanged. In Figure 3, western blot analysis of protein captured in the EGF Receptor mouse mAb coated microwell shows a single band corresponding to the EGF Receptor. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.