Equipamentos para Laboratórios | Equipamento para Laboratório

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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity: H

Reactivity Key:  H=Human

Description

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 #2223* 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 #2257* 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. 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.

Specificity / Sensitivity

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.

Western Blotting

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.

Sandwich ELISA

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.

Sandwich ELISA

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 capture Ab #2223. 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 EGF Receptor Ab #2232. A single band corresponding to the EGF Receptor is detected in the captured material (lane 2).

Background

The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling 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 c-Cbl, an adaptor protein that leads 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 residues (Tyr1148 and Tyr1173) provides 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 to 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.

Protocols

7250:  Sandwich ELISA

Companion Products

    * 7074 Anti-rabbit IgG, HRP-linked Antibody
    * 9803 Cell Lysis Buffer (10X)
    * 7187 PathScan® Phospho-EGF Receptor (Tyr1173) Sandwich ELISA Kit
    * 7189 PathScan® Phospho-EGF Receptor (Tyr845) Sandwich ELISA Kit
    * 7240 PathScan® Phospho-EGF Receptor (Tyr1068) Sandwich ELISA Kit