03/16/2023
Isolation and enrichment of Golgi Bodies from rice seedlings
Kazusato Oikawa, Eppendorf Lab for Biomaterial Chemistry, Kyoto
Shuichi Kani, Eppendorf Himac Technologies, Tokyo
Mark Hünken, Eppendorf SE
The Golgi apparatus of eukaryotic cells was first described more than 120 years ago by Camillo Golgi. Advances in (electron) microscopy revealed the complex structure while further biochemical analysis enlighted various functions of this organelle within the cell [1]. In cells of higher organisms, the Golgi apparatus is responsible for the synthesis of complex polysaccharides and the processing and distribution of proteins to other organelles as part of the secretory pathway [2].
One example of such a protein is α-amylase, a glycosidase responsible for the hydrolysis of starch molecules within plants. It was shown that α-amylase is synthesized at the endoplasmic reticulum (ER) ribosomes, glycosylated within the ER-lumen, and then transported into the Golgi apparatus for oligosaccharide modification [3].
However, as the Golgi apparatus forms a complex structure with other membrane systems like the endoplasmic reticulum (ER) [1], it is particularly difficult to isolate distinct parts of this organelle. Indeed, fractions of Golgi membranes are often contaminated with parts of other connected membrane systems like the vacuole [1]. Density gradient centrifugation is one of the most established techniques used for the enrichment of specific membranes [1].
In this Application Note, we describe a technique using a sequence of differential pelleting and density gradient centrifugation to obtain fractions of Golgi apparatus membranes from rice seedlings. The applied technique allows to gain extracts of high purity and quality for further downstream analysis like mass spectrometry.