Takasago T, Nakamura K, Kashiwagi S, Inoue S, Ito H, Takeo K
Department of Neurosurgery, Yamaguchi University School of Medicine, Japan.
Electrophoresis. 1992 Jun;13(6):373-8. doi: 10.1002/elps.1150130176.
A new method of type III collagen analysis by uninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with immunoblotting was developed. The electrophoresis was carried out with gels containing 4 M urea. A negatively charged reducing agent, thioglycolic acid, was added to the running buffer of the cathodic reservoir between 15 and 20 min after Bromphenol Blue (BPB) migrated to the top of the separating gel, to reduce interchain disulfide binding of the collagen. The polymorphic type III collagens, i.e., an alpha-chain derived from a trimer [alpha 1(III)]3 with interchain disulfide bonds but without covalent cross-links, alpha 1(III), a beta-chain with covalent cross-links, beta(III), or an alpha-chain released from a trimer without reduction of the disulfide bonds, alpha*1(III), were identified by immunostaining and quantified by densitometry. Using this method, changes in collagen type III polymorphism with aging were examined in the aorta, brachial artery, and skin of rats. The total quantity of collagen type III decreased with aging in all tissues. beta(III) was the major component in the aorta and brachial artery, but alpha 1(III) was the major component in the skin. With increasing age from 3 to 60 weeks, the ratio of beta(III) to alpha 1(III), which is correlated with the extent of covalent cross-linking, showed a steep increase in the aorta but only a slight increase in the skin and it remained almost constant in the brachial artery.(ABSTRACT TRUNCATED AT 250 WORDS)
开发了一种通过不间断十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结合免疫印迹法分析III型胶原蛋白的新方法。电泳在含有4M尿素的凝胶上进行。在溴酚蓝（BPB）迁移至分离胶顶部后15至20分钟之间，向阴极槽的运行缓冲液中加入带负电荷的还原剂巯基乙酸，以减少胶原蛋白的链间二硫键结合。通过免疫染色鉴定并通过光密度法对多态性III型胶原蛋白进行定量，即源自三聚体[α1(III)]3、具有链间二硫键但无共价交联的α链、α1(III)、具有共价交联的β链、β(III)或未还原二硫键而从三聚体释放的α链、α*1(III)。使用该方法，研究了大鼠主动脉、肱动脉和皮肤中III型胶原蛋白多态性随衰老的变化。所有组织中III型胶原蛋白的总量均随衰老而减少。β(III)是主动脉和肱动脉中的主要成分，但α1(III)是皮肤中的主要成分。从3周到60周龄，与共价交联程度相关的β(III)与α1(III)的比例在主动脉中急剧增加，但在皮肤中仅略有增加，在肱动脉中几乎保持不变。（摘要截短于250字）
