STUDYING EGFR INTERACTING PARTNERS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 EGFR（表皮生长因子受体）是细胞外蛋白配体的表皮生长因子家族（EGF家族）成员的细胞表面受体，通过与表皮生长因子和转化生长因子（TGF）结合而被激活。导致EGFR过表达的突变与许多癌症相关，包括肺癌和多形性胶质母细胞瘤。为了分离EGFR及其相互作用伙伴，我们测试了几种市售的单克隆和多克隆抗EGFR抗体。然而，这些方法均未成功用于免疫隔离。因此，我们继续测试内部开发的抗体（兔多克隆），我们必须进一步纯化以获得可用于免疫分离的抗体。经过仔细优化的裂解缓冲液，并测试磁珠与琼脂糖珠，我们现在已经分离出EGFR与相关蛋白。我们的研究结果证实了以前已知的相互作用伙伴，如GRB 2，并指出EGFR上的许多磷酸化位点。为了获得尽可能清楚的背景（非特异性结合）免疫分离，我们在免疫分离前检测了过滤步骤的使用。过滤后，我们仍然可以检测到EGFR和GRB 2。在成功分离EGFR后，我们现在开始比较野生型和两种突变型EGFR蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. EGFR (the epidermal growth factor receptor) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands, being activated by binding to epidermal growth factor and transforming growth factor ¿ (TGF¿). Mutations that lead to EGFR overexpression have been associated with a number of cancers, including lung cancer and glioblastoma multiforme. To isolate EGFR with its interacting partners we tested several monoclonal and polyclonal commercially available anti-EGFR antibodies. However, none of these proved successful for immunoisolations. We therefore proceeded in testing in-house-developed antibodies (rabbit polyclonal), which we had to further purify to achieve antibodies usable for immunoisolations. After careful optimization of lysis buffers, and tests of magnetic beads versus agarose beads, we now have isolated EGFR with associated proteins. Our results confirmed previous known interacting partners, such as GRB2, and indicated numerous phosphorylation sites on EGFR. In our effort to obtain immunoisolations as clear as possible of background (non-specific binding), we tested the use of a filtration step prior to immunoisolations. Following filtration, we could still detect EGFR and GRB2. Having successfully isolated EGFR, we have now started the comparison between wild type and two mutant EGFR proteins.