A novel method to isolate and map endothelial membrane proteins from pulmonary vasculature.
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.
Zhou
Ying-Jiang
YJ
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Univ., 22 Hankou Rd., Nanjing 210093, China.
Wang
Shi-Quan
SQ
Zhang
Jing
J
Zhang
Wei
W
Bi
Feng
F
Guo
Zhi-Gang
ZG
Ding
Bi-Sen
BS
Kumar
Pat
P
Liu
Jian-Ning
JN
Tan
Xiang-Yang
XY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D005260:Female; D015151:Immunoblotting; D007150:Immunohistochemistry; D008168:Lung / Q000098:blood supply*; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins