Overview:
The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase. Known ligands of EGFR include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding induces receptor homo- and hetero-dimerization and tyrosine autophosphorylation, which triggers downstream signaling events. The consequences of EFGR signaling include cell proliferation, differentiation, motility, and cell survival (1). Activation of EGFR triggers mitogenic signaling in gastrointestinal mucosa and EGFR upregulated in colon cancers and most neoplasms (2). EGFR stimulation also activates the ERK-signaling pathway in normal gastric epithelial and colon cancer cell lines. In contrast, selective inhibition of EGFR significantly reduces ERK2 activation, c-fos mRNA expression and cell proliferation. Mutations in EGFR are also implicated in specific forms of lung cancer.
References:
1. Wang K, et al: Epidermal growth factor receptor-deficient mice have delayed primary endochondral ossification because of defective osteoclast recruitment. J. Biol. Chem. 279: 53848-53856, 2004. 2. Kobayashi S, et al: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. New Eng. J. Med. 352: 786-792, 2005.
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(1)确定抗体的名字,注意中英文名字、别名、亚型等信息。
(2)确定您的实验类型,Elisa,WB,IHC,ICC,或者是FACS。每种抗体说明书都会列出抗体经验证过适用的实验类型,如果抗体说明书没有提及的应用类型,并不意味着该抗体不适用于此种分析应用类型,而仅是说明尚未经过此种实验验证。
(3)确定实验样本的种属,Human,Mouse,Rat等。我们的抗体说明书都列出该抗体验证过的适用物种,可根据说明书列出已验证的种属来选择适合你实验的抗体。
(4)样本抗原蛋白的结构性质。了解样本抗原蛋白的结构性质有助于选择最合适的抗体,待测样本蛋白的结构域和样本在提取和处理过程中是否会变性,蛋白空间构象的改变,会影响抗体的免疫亲和反应。
(5)单多克隆抗体的选择。一般单克隆抗体特异性强,但亲和力相对小,检测抗原灵敏度相对就低;而多克隆抗体特异性稍弱,但抗体的亲和力强,灵敏度高,但易出现非特异性染色(可以通过封闭等避免)。
2. 二抗选择
(1)种属来源。主要根据一抗种属来源而决定二抗来源,如一抗是小鼠来源,那二抗就买抗小鼠的即可(羊、兔等均可)。
(2)标记物的选择。有HRP、Biotin、荧光素等标记物。免疫组化,WB二抗主要选择HRP,Biotin标记的二抗,而免疫荧光染色可按实验需要选择不同荧光素标记的二抗,如FITC、Cy3、PE等。
不过也有用 HRP标记的protein a或g的,前提是你跑的样里没有IgG之类的东西。

