King B J, Lee L S, Rackemann R G, Scott P T
University of Central Queensland, Rockhampton, Australia.
J Biochem Biophys Methods. 1994 Dec;29(3-4):295-305. doi: 10.1016/0165-022x(94)90040-x.
Extracting proteins from vegetative tissues while maintaining good enzyme activity and electrophoretic resolution presents numerous problems due to the presence of phenols, quinones, proteases and other components released during cell disruption. To overcome this problem in Citrus leaves, an extraction buffer was developed which contained EDTA, potassium chloride, magnesium chloride hexahydrate, PVP-40, 2-mercaptoethanol and bovine serum albumin in a Tris-HCl buffer, pH 7.5. This extraction buffer was used in association with liquid nitrogen for sample preparation. Buffers used in previous studies for Citrus isozyme extraction for PAGE were found to provide unsatisfactory resolution and activity for the three enzyme systems investigated (malate dehydrogenase, 6-phosphogluconate dehydrogenase and shikimate dehydrogenase). This extraction buffer maintains high enzyme activity and provides good resolution in PAGE gels suitable for densitometric analysis.
从营养组织中提取蛋白质并同时保持良好的酶活性和电泳分辨率存在诸多问题，这是因为细胞破碎过程中会释放出酚类、醌类、蛋白酶及其他成分。为解决柑橘叶片中的这一问题，研发了一种提取缓冲液，其在pH 7.5的Tris-HCl缓冲液中含有EDTA、氯化钾、六水合氯化镁、聚乙烯吡咯烷酮-40、2-巯基乙醇和牛血清白蛋白。该提取缓冲液与液氮联合用于样品制备。此前用于柑橘同工酶提取以进行聚丙烯酰胺凝胶电泳（PAGE）的缓冲液，对于所研究的三种酶系统（苹果酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和莽草酸脱氢酶）而言，分辨率和活性均不尽人意。这种提取缓冲液能保持较高的酶活性，并在适用于光密度分析的PAGE凝胶中提供良好的分辨率。
