Figure 1. The relationship between protein concentration of lysates from A-431 cells, untreated or treated with hEGF #8916, and immediate light generation with chemiluminescent substrate. After starvation, A-431 cells (85% confluence) were treated with EGF (100 ng/ml, 5 min at 37°C) and then lysed. Graph inset corresponding to the shaded area shows high sensitivity and a linear response at the low protein concentration range.
Figure 2. The relationship between protein concentration of lysates prepared using H1975 cells, lysed with (phospho) and without (nonphospho) the addition of phosphatase inhibitors to the lysis buffer, and immediate light generation using chemiluminescent substrate.
|Product Includes||Volume||Solution Color|
|EGF Receptor Mouse mAb Coated Microwells||96 tests|
|Phospho-EGF Receptor (Tyr1068) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||5.5 ml||Green|
|HRP Diluent||5.5 ml||Red|
|Luminol/Enhancer Solution||3 ml|
|Stable Peroxide Buffer||3 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|
CST's PathScan® Phospho-EGF Receptor (Tyr1068) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-EGF Receptor (Tyr1068) protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. An EGF Receptor Mouse mAb has been coated onto the microwells. After incubation with cell lysates, EGF receptor proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, Phospho-EGF Receptor (Tyr1068) Rabbit mAb is added to detect the captured phospho-EGF Receptor protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of Phospho-EGF Receptor (Tyr1068).
Antibodies in kit are custom formulations specific to kit.
NOTE: Refer to product-specific datasheets for assay incubation temperature. This chemiluminescent ELISA is offered in low volume microplates. Only 50 µl of samples or reagents are required in each microwell.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
posted November 2013
Protocol Id: 66
PathScan® Phospho-EGF Receptor (Tyr1068) Chemiluminescent Sandwich ELISA Kit #7036 detects endogenous levels of phospho-EGF Receptor (Tyr1068) protein in human cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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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