Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-EGF Receptor (Tyr1068) (E746-A750del Specific) Sandwich ELISA Kit #7702

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Kit Includes Volume Solution Color
EGF Receptor (E746-A750del Specific) Rabbit Antibody coated microwell 96 tests
Phospho-EGF Receptor (Tyr1068) Mouse Detection Antibody 11 ml Green
Anti-rabbit IgG, HRP- linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 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

The PathScan® Phospho-EGF Receptor (Tyr1068) (E746-A750del Specific) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-EGF receptor (Tyr1068) (E746-A750del) mutant protein. An EGF Receptor (E746-A750del Specific) Rabbit Antibody has been coated on the microwells. After incubation with cell lysates, EGF receptor (E746-A750del) mutant protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-EGF Receptor (Tyr1068) Mouse Detection Antibody is added to detect captured phosphorylated EGF receptor (E746-A750del) mutant protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-EGF receptor (Tyr1068) (E746-A750del) mutant protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-EGF Receptor (Tyr1068) (E746-A750del Specific) Sandwich ELISA Kit #7702 detects endogenous levels of phospho-EGF receptor (Tyr1068) (E746-A750del) mutant protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Constitutive phosphorylation of EGF receptor (E746-A750del) mutant protein in HCC827 cells lysed in the presence of phosphatase inhibitors* (phospho lysates) is detected by the PathScan® Phospho-EGF Receptor (Tyr1068) (E746-A750del Specific) Sandwich ELISA Kit #7702. In contrast, a low level of phospho-EGF receptor (Tyr1068) (E746-A750del) protein is detected in HCC827 cells lysed in the absence of phosphatase inhibitors* (nonphospho lysates). This kit does not detect wild type EGFR protein (phospho or nonphospho) from A-431 cells or EGFR (L858R) mutant protein (phospho or nonphospho) from H3255 cells. Absorbance at 450 nm is shown in the top figure (positive signal is seen only in HCC827 phospho lysates), while western blots using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate, and Na3VO4.

Sensitivity

Sensitivity

Figure 2. The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm is shown. HCC827 cells were cultured (85% confluence) and lysed with or without the addition of phosphatase inhibitor to the lysis buffer (phospho or nonphospho lysate, respectively).

Background

The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Research studies have shown that somatic mutations in the tyrosine kinase domain of EGF receptor (EGFR) are present in a subset of lung adenocarinomas that respond to EGFR inhibitors, such as gefinitib and erlotinib (1-3). Two types of mutations account for approximately 90% of mutated cases: a specific point mutation, L858R, that occurs in exon 21 and short in-frame deletions in exon 19 (4,5). The most frequent exon 19 deletion is E746-A750, accounting for 90% of lesions at this site, although some rare variants occur.

  1. Lynch, T.J. et al. (2004) N Engl J Med 350, 2129-39.
  2. Pao, W. et al. (2004) Proc Natl Acad Sci USA 101, 13306-11.
  3. Haber, D.A. et al. (2005) Cold Spring Harb Symp Quant Biol 70, 419-26.
  4. Kosaka, T. et al. (2004) Cancer Res 64, 8919-23.
  5. Riely, G.J. et al. (2006) Clin Cancer Res 12, 7232-41.

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