Preparation of extracts for electrophoresis from citrus leaves

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.