Boman B M, Lovas S, Abraham C, Adrian T E, Murphy R F, Marbello R, Bhattacharya G
Creighton Cancer Center, Creighton University School of Medicine, Omaha, Nebraska 68178.
Biochem Biophys Res Commun. 1995 Jan 26;206(3):909-15. doi: 10.1006/bbrc.1995.1129.
A radioimmunoassay (RIA) has been developed for the adenomatous polyposis coli protein (APC). High-avidity rabbit polyclonal antibodies were produced against synthetic peptides corresponding to amino acids 1865-1881 (APC-1) and to amino acids 1336-1350 (APC-2) in APC's 2844 amino acid sequence. Both antibodies were utilized in RIA to evaluate full-length APC that is present in the insoluble particulate fraction of cell lysates. High salt extraction, often employed for coiled-coil type protein preparations, was found to be useful for extraction of APC from lysates of normal colonic epithelium. Proteolytic digestion of high salt extracts increased antibody reactivity toward both epitopes, suggesting that APC-1 and APC-2 antigenic sites are partially concealed due to APC's involvement in multiprotein complexes. Thus, RIA using our antibodies will provide a valuable tool for APC protein purification and in studies for elucidating APC's biologic function.
已开发出一种针对腺瘤性息肉病大肠杆菌蛋白（APC）的放射免疫分析方法（RIA）。针对APC 2844个氨基酸序列中对应于1865 - 1881位氨基酸（APC - 1）和1336 - 1350位氨基酸（APC - 2）的合成肽，制备了高亲和力兔多克隆抗体。两种抗体均用于RIA，以评估存在于细胞裂解物不溶性颗粒部分中的全长APC。高盐提取常用于卷曲螺旋型蛋白质的制备，发现其可用于从正常结肠上皮细胞裂解物中提取APC。高盐提取物的蛋白酶消化增加了抗体对两个表位的反应性，表明由于APC参与多蛋白复合物，APC - 1和APC - 2抗原位点被部分掩盖。因此，使用我们的抗体进行的RIA将为APC蛋白纯化以及阐明APC生物学功能的研究提供有价值的工具。
