Human Phospho-EGFR DuoSet IC ELISA

Figure 1: The Human Phospho-EGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis The human epidermoid carcinoma cell line, A431, was treated with 25 ng/mL recombinant human EGF (R&D Systems Catalog #236-EG) for 5 minutes to induce tyrosine phosphorylation of EGF R. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an anti-EGF R monoclonal antibody and anti-mouse IgG agarose. Immunoblots were incubated with an HRP-conjugated anti-phospho-tyrosine monoclonal antibody (R&D Systems, Catalog #HAM1676) to detect phosphor-EGF R. Bands were visualized by chemiluminescent detection. Human Phospho-EGF R can be detected in this DuoSet IC ELISA by using approximately 2-4 times less lysate than is needed for a conventional IP-Western blot.

Figure 2: The Human Phospho-EGF R DuoSet IC ELISA detects ligand-induced EGF R tyrosine phosphorylation A431 cells were untreated or treated with 25 ng/mL recombinant human EGF for 5 minutes. ELISA and IP-Western blot (inset) analyses were done using 25 and 50 μg of lysate, respectively. IP-Western blots for phospho-EGF R (p-EGF R) were done as described in Figure 1. Blots were stripped and total EGF R was detected using a biotinylated anti-EGF R polyclonal antibody (R&D Systems, Catalog #AF231).

Figure 3. The quantification of Human Phospho-EGF R DuoSet IC ELISA is demonstrated by using cells pretreated with the inhibitor PD168393 A431 cells were incubated with no additions or treated with 25 ng/mL recombinant human EGF for 5 minutes, after either being treated with or without 1 nM, 10 nM, or 100 nM PD168393. ELISA and IP-Western blot (inset) analyses were done using 25 and 50 μg of lysate, respectively. IP-Western blots for phospho-EGF R (p-EGF R) were done as described in Figure 1. Total EGF R blots were done as described in Figure 2. The DuoSet IC ELISA results correlate well with the relative amount of phosphorylated EGF R detected by IP-Western blot.

Figure 4. The specificity of Human Phospho-EGF R DuoSet IC ELISA is confirmed by receptor competition A431 cells were treated with 25 ng/mL recombinant human EGF for 5 minutes. The indicated amounts of recombinant extracellular domains of human EGF R (R&D Systems, Catalog #1095-ER), human ErbB2/Fc Chimera (R&D Systems, Catalog #1129-ER), human ErbB3/Fc Chimera (R&D Systems, Catalog #348-RB), or human ErbB4/Fc Chimera (R&D Systems, Catalog #1131-ER) were added to 25 μg of lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human EGF R.