Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-EGF Receptor (Tyr845) Sandwich ELISA Kit #7189

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Kit Includes Volume Solution Color
EGF Receptor mAb coated microwells 96 tests
P-EGF Receptor (Y845)Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
Sample Diluent 25 ml Blue
Sealing Tape 2 sheets
Cell Lysis Buffer (10X) #9803 15 ml

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

CST's PathScan® Phospho-EGF Receptor (Tyr845) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-EGF Receptor (Tyr845) protein. A 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, Phospho-EGF Receptor (Tyr845) Rabbit mAb is added to detect the captured phospho-EGF Receptor protein. Anti-rabbit IgG, HRP-linked Antibody #7074* 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 phospho-EGF Receptor (Tyr845) protein.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-EGF Receptor (Tyr845) Sandwich ELISA Kit detects endogenous levels of phospho-EGF Receptor (Tyr845) protein. As shown in figure 1, using the Phospho-EGF Receptor (Tyr845) ELISA Kit #7189, a significant induction of Phospho-EGF Receptor (Tyr845) in A-431 cells treated with EGF is detected. However, the levels of EGF Receptor (phospho and non-phospho) detected by PathScan® Total EGF Receptor Sandwich ELISA Kit #7250, remain unchanged. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Western Blotting

Western Blotting

Figure 1: Treatment of A431 cells with EGF stimulates phosphorylation of EGF Receptor at Tyr845, detected by PathScan® Phospho-EGF Receptor (Tyr845) Sandwich ELISA kit #7189, but does not affect the level of total EGF Receptor detected by PathScan® Total EGF Receptor Sandwich ELISA kit #7250. OD 450 nm readings are shown in the top figure, while the corresponding Western blot using Phospho-EGF Receptor (Tyr845) Antibody #2231 (right panel) or EGF Receptor Antibody #2232 (left panel), is shown in the bottom figure.

Sandwich ELISA

Sandwich ELISA

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.

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.

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