A quantitative dot-blot immunoassay for integral membrane proteins: preparation of pancreatic plasma membranes containing apical and basolateral domains.
A quantitative dot-blot immunoassay for integral membrane proteins: preparation of pancreatic plasma membranes containing apical and basolateral domains.
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完整膜蛋白的定量斑点印迹免疫分析：含有顶端和基底外侧结构域的胰腺质膜的制备。
DOI：
10.1016/0003-2697(91)90173-q
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发表时间：
1991
影响因子：
2.9
通讯作者：
DeLisle,RC
中科院分区：
文献类型：
--
作者：
DeLisle,RC
对标准斑点印迹免疫测定技术进行了修改，用于定量膜蛋白抗原。该修饰涉及信号产生步骤，其通过使用固体支持物（硝化纤维素）的冲出的威尔斯孔来进行，以产生可溶性有色反应产物。这种方法避免了不溶性反应产物的不均匀性以及当通过光密度测定法扫描这种产物时遇到的随后的定量问题。该方法进行了验证，通过比较的纯化和总回收率的一个已知的质膜酶标记物与完整的膜抗原的定义的定位在亚细胞分级分离的小鼠胰腺。最终的质膜部分含有基底外侧（富集20倍）和顶膜（富集24倍）结构域。
Modifications of standard dot-blot immunoassay techniques have been made for the quantitation of integral membrane protein antigens. The modification involves the signal development step, which is performed by using punched-out wells of the solid support (nitrocellulose) to yield a soluble colored reaction product. This approach avoids the inhomogeneity of an insoluble reaction product and the subsequent quantitation problems encountered when such product is scanned by densitometry. The method was validated by comparing the purification and overall recoveries of a known plasma membrane enzymatic marker with integral membrane antigens of defined localization during subcellular fractionation of mouse pancreas. The final plasma membrane fraction contained both basolateral (∼20-fold enriched) and apical membrane (∼4-fold enriched) domains.