In 1992, Margolis and colleagues published a paper in the journal Nature that explored the interaction between EGFR and a newly discovered protein called Vav For their experiments, the authors expressed Vav and EGFR in cultured cells that were either stimulated with EGF or mock-treated (no EGF). Next, they immunoprecipitated Vav from the cell lysates using anti-Vav antibodies and ran the immunoprecipitates on SDS-PAGE gels. Next, they did immunoblots to detect either phospho-tyrosine (P-Tyr) or EGFR. One of their figures is reproduced below IP Ab: anti-Vav Legend: EGF: M, (K) 191- P Ab- immunoprecipitation antibody; the antibody used to pull protein complexes out of the cell lysate. Blot Ab-Blot antibody: the primary antibody that was used on the immunoblot, “anti” refers to the protein or post-translational modification that the antibody will bind to. EGFR Epidermal growth factor receptor; a receptor tyrosine kinase (RTK) EGF Epidermal growth factor; the ligand for the RTK, 92 EGF (+1-): was EGF added to the cells, yes or no? Blot Ab: anti-P-Tyr anti-EGFR (0-Molecular mass of a protein nk 1. Which lane contains a band representing Vav only? 2. Which lane contains a band representing EGFR only? 3. Which protein is bigger, Vav or EGFR? 4. In this experiment using cultured cells, is stimulation with EGF required for: a. Vav phosphorylation? b. EGFR phosphorylation? c. Binding between Vav and EGFR? 5. Why is there no band in lane 3?
