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.