STUDYING EGFR INTERACTING PARTNERS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 EGFR(表皮生长因子受体)是细胞外蛋白配体中表皮生长因子家族(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)结合而激活。导致EGFR过表达的突变与许多癌症有关，包括肺癌和多形性胶质母细胞瘤。为了分离EGFR及其相互作用的伙伴，我们测试了几种商用的抗EGFR的单克隆和多克隆抗体。然而，事实证明，这些方法都不能用于免疫隔离。因此，我们进行了内部开发的抗体(兔多克隆)的测试，我们必须进一步纯化这些抗体，以获得可用于免疫分离的抗体。经过对裂解缓冲液的仔细优化，以及磁珠和琼脂糖珠的测试，我们现在已经分离出带有相关蛋白的EGFR。我们的结果证实了以前已知的相互作用伙伴，如Grb2，并指出了EGFR上的大量磷酸化位点。为了获得尽可能清晰的免疫分离背景(非特异性结合)，我们在免疫分离之前测试了过滤步骤的使用。过滤后仍可检测到EGFR和Grb2。在成功分离EGFR后，我们现在开始比较野生型和两个突变的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.