Immunoblotting of hydrophobic integral membrane proteins.
Immunoblotting of hydrophobic integral membrane proteins.
复制标题
疏水性整合膜蛋白的免疫印迹。
DOI：
10.1016/0003-2697(88)90304-1
复制
发表时间：
1988
影响因子：
2.9
通讯作者：
Lazarow,PB
中科院分区：
文献类型：
--
作者：
Small,GM;Imanaka,T;Lazarow,PB
出于诊断和研究目的，经常需要通过免疫印迹法测量复杂混合物（如组织活检匀浆）中的少量蛋白质。对于可溶性蛋白质给出优异结果的标准免疫印迹程序对于一些疏水膜蛋白出乎意料地给出低且不可再现的信号。我们发现，这是由于效率低下的电泳转移到硝酸纤维素，这可以通过修改transblot缓冲液。过氧化物酶体以及其他大鼠和人类肝脏蛋白的疏水性整合膜蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，并转移到硝酸纤维素过滤器。硝化纤维素结合的蛋白质通过染色和免疫印迹检测抗过氧化物酶体的22 kDa的膜蛋白加上125 I标记的蛋白A的抗血清。用0.7 m甘氨酸和25 mm Tris（pH7.7）但不含甲醇的改良transblot缓冲液，可以使用短得多的转移时间，并显著改善膜蛋白的电泳转移，从而可以在人肝活检组织匀浆中检测到过氧化物酶体整合膜蛋白。
For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus125I-labeled protein A. A modified transblot buffer with 0.7 m glycine and 25 mm Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be casily detected in human liver biopsy homogenates.