Biochemical sub-fractionation of the mammalian Golgi apparatus.
We have exploited the breakdown of the Golgi apparatus that occurs during mitosis to isolate subfractions using immuno-affinity methods. Rat liver Golgi stacks were treated with mitotic cytosol from HeLa cells, and the fragments were then incubated with antibodies immobilized on magnetic beads. Antibodies against the cis-Golgi marker, GM130, bound membranes that were depleted in the trans-Golgi network marker, TGN38, whereas antibodies against the cytoplasmic tail of TGN38 did the reverse. A range of other Golgi enzymes, SNAREs and tethers were also tested and were found to bind to anti-GM130 antibodies to an extent that reflected their proximity to cis-cisternae as determined by other techniques. This method should provide a useful complement to the immuno-EM methods presently used to map the Golgi apparatus.
Taguchi
Tomohiko
T
Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8002, USA.
Pypaert
Marc
M
Warren
Graham
G
D000818:Animals; D015415:Biomarkers; D002458:Cell Fractionation / Q000379:methods*; D006056:Golgi Apparatus / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008099:Liver / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D008938:Mitosis / Q000502:physiology; D051381:Rats
D015415:Biomarkers