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Biochemical Sub‐Fractionation of the Mammalian Golgi Apparatus
Author(s) -
Taguchi Tomohiko,
Pypaert Marc,
Warren Graham
Publication year - 2003
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2003.00091.x
Subject(s) - golgi apparatus , biology , microbiology and biotechnology , mitosis , cytoplasm , cell fractionation , endoplasmic reticulum , vesicle , cytosol , antibody , golgi membrane , biochemistry , membrane , enzyme , immunology
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 .