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Am J Physiol Cell Physiol 288: C950-C956, 2005. First published December 8, 2004; doi:10.1152/ajpcell.00262.2004
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Methods in Cell Physiology

A novel method to isolate and map endothelial membrane proteins from pulmonary vasculature

Ying-Jiang Zhou,1 Shi-Quan Wang,1 Jing Zhang,1 Wei Zhang,1 Feng Bi,1 Zhi-Gang Guo,1 Bi-Sen Ding,1 Pat Kumar,2 Jian-Ning Liu,1 and Xiang-Yang Tan1

1Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China; and 2Department of Biological Sciences, Manchester Metropolitan Unviersity, Manchester, United Kingdom

Submitted 1 June 2004 ; accepted in final form 8 December 2004

Vascular endothelium has attracted extensive attention due to its important role in many physiological and pathological processes. Many methods have been developed to study the components and their functions in vascular endothelium. Here we report a novel approach to investigate vascular endothelium using normal rat lungs as the model. We perfused lung vascular beds with sulfosuccinimidyl-6-(biotinamido) hexanoate, a biotin analog, to label endothelial membrane proteins. The biotinylated proteins were isolated from lung homogenate with immobilized monomeric avidin and confirmed to be highly pure endothelial membrane proteins with little contamination of intracellular proteins. These biotinylated proteins were used as immunogens for development of monoclonal antibodies. Indeed, newly generated monoclonal antibodies have revealed different expression patterns of proteins across tissues. Some proteins were found highly specifically expressed to capillary vessels of pulmonary vasculature. This method has also been proven useful for investigating vasculature of other organs, as this study explored.

vascular endothelium; biotinylation; tissue specific; monoclonal antibodies


Address for reprint requests and other correspondence: J.-N. Liu or X.-Y. Tan, Institute of Molecular Medicine, Nanjing Univ., 22 Hankou Rd., Nanjing 210093, China (E-mail: jnliu{at}verizon.net or xiangyangtan{at}yahoo.com)






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