Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total EGF Receptor Chemiluminescent Sandwich ELISA Kit #7297

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
EGF Receptor Mouse mAb Coated Microwells 96 tests
EGF Receptor Rabbit Detection Antibody 5.5 ml Green
Anti-rabbit IgG, HRP-Linked Ab 5.5 ml Red
Luminol/Enhancer Solution 3 ml Colorless
Stable Peroxide Buffer 3 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

PathScan® Total EGF Receptor Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total EGF receptor protein. 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, both phospho- and nonphospho-EGF receptor proteins are captured by the coated antibody. Following extensive washing, EGF Receptor Rabbit mAb is added to detect both the captured phospho- and nonphospho-EGF receptor protein. Anti-rabbit IgG, HRP-linked Antibody #7074 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 total EGF receptor protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Total EGF Receptor Chemiluminescent Sandwich ELISA Kit #7297 detects endogenous levels of EGF receptor protein. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

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 hEGF (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.

ELISA

ELISA

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.

Background

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).

  1. Hackel, P.O. et al. (1999) Curr Opin Cell Biol 11, 184-9.
  2. Zwick, E. et al. (1999) Trends Pharmacol Sci 20, 408-12.
  3. Cooper, J.A. and Howell, B. (1993) Cell 73, 1051-4.
  4. Hubbard, S.R. et al. (1994) Nature 372, 746-54.
  5. Biscardi, J.S. et al. (1999) J Biol Chem 274, 8335-43.
  6. Emlet, D.R. et al. (1997) J Biol Chem 272, 4079-86.
  7. Levkowitz, G. et al. (1999) Mol Cell 4, 1029-40.
  8. Ettenberg, S.A. et al. (1999) Oncogene 18, 1855-66.
  9. Rojas, M. et al. (1996) J Biol Chem 271, 27456-61.
  10. Feinmesser, R.L. et al. (1999) J Biol Chem 274, 16168-73.

Application References

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Protocols

* Product-specific protocol.

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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