Human Total EGFR DuoSet IC ELISA

R&D Systems | Catalog # DYC1854-2

R&D Systems
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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

31.2-2000 pg/mL

Sample Type

Cell Lysates

Reactivity

Human

Human Total EGFR DuoSet IC ELISA Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Available in 2, 5, and 15- (96-well) plate pack sizes
  • Economical alternative to Western blot
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Product Summary for Human Total EGFR DuoSet IC ELISA

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total EGFR in cell lysates. An immobilized capture antibody specific for EGFR binds both phosphorylated and unphosphorylated EGFR. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

Cell lysates (100 µL)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Human Total EGFR DuoSet IC ELISA

Standard Curve

Figure 1. The Human Total-EGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis

Lysates prepared from the human epidermoid carcinoma cell line, A431, were diluted in series and analyzed by (A) IP-Western blot and (B) R&D Systems’ Human Total-EGF R DuoSet IC ELISA (Catalog # DYC1854). IPs were performed using anti-EGF R polyclonal antibody (Catalog # AF231) Immunoblots were incubated with a biotinylated anti-EGF R polyclonal antibody (Catalog # BAF231) to detect total EGF R. Blots were incubated with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Human EGF R can be detected by the Human Total-EGF R DuoSet IC ELISA by using approximately 200 to 500 times less lysate than is needed for a conventional IP-Western blot.

Figure 2. The Human Total-EGF R DuoSet IC ELISA measures the relative level of EGF R

Lysates were prepared from A431 cells, human immortalized fibroblast cells (CCD1070SK), and human breast adenocarcinoma cells (SkBr3). ELISA analysis was done using 125 ng (A431) and 2000 ng (CCD1070SK and SkBr3) of lysate. IP-Western blot analysis (inset) was done using 5 μg (A431) and 100 μg (CCD1070SK and SkBr3) of lysate. The IP-Western blot was performed as described in Figure 1. The smaller band visible in IP-Western blot is an immature EGF R glycosylation intermediate.

Kit Contents for Human Total EGFR DuoSet IC ELISA

  • Capture Antibody
  • Conjugated Detection Antibody
  • Calibrated Immunoassay Standard or Control
  • Streptavidin-HRP

Other Reagents Required


PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Lysis Buffer*

IC Diluent*

Blocking Buffer*

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent

*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: EGFR

EGFR (epidermal growth factor receptor), also known as HER-1, ErbB1, or ErbB, is a transmembrane receptor tyrosine kinase that binds a subset of the EGF family ligands including EGF, Amphiregulin, TGF-alpha, Betacellulin, Epiregulin, HB-EGF, and Epigen. Ligand binding induces EGFR homodimerization as well as heterdimerization with ErbB2, resulting in kinase activation, tyrosine phosphorylation and cell signaling. EGFR can also be recruited to form heterodimers with the ligand-activated ErbB3 or ErbB4. EGFR signaling regulates multiple biological functions including cell proliferation, differentiation, motility, and apoptosis. EGFR is overexpressed in a wide variety of tumors and is the target of several anti-cancer drugs. Soluble receptors consisting of the extracellular ligand binding domain are generated by alternate splicing in human and mouse.

Long Name

Epidermal Growth Factor Receptor

Alternate Names

EGF R, ErbB, ErbB1, HER-1

Entrez Gene IDs

1956 (Human); 13649 (Mouse); 24329 (Rat); 102138724 (Cynomolgus Monkey)

Gene Symbol

EGFR

Additional EGFR Products

Product Documents for Human Total EGFR DuoSet IC ELISA

Certificate of Analysis

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Product Specific Notices for Human Total EGFR DuoSet IC ELISA

For research use only

Citations for Human Total EGFR DuoSet IC ELISA

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  • Human Total EGFR DuoSet IC ELISA
    Name: Balaji Mahender
    Sample Tested: Cell culture supernatant
    Verified Customer | Posted 01/18/2018

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