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. Anti...

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Bibliographic Details
Published inTraffic (Copenhagen, Denmark) Vol. 4; no. 5; pp. 344 - 352
Main Authors Taguchi, Tomohiko, Pypaert, Marc, Warren, Graham
Format Journal Article
LanguageEnglish
Published Oxford, UK Blackwell Publishing Ltd 01.05.2003
Subjects
Animals
Biomarkers
Cell Fractionation - methods
fractionation
Golgi
Golgi Apparatus - chemistry
Golgi Apparatus - immunology
Golgi Apparatus - ultrastructure
golgin
HeLa Cells
Humans
Immunoassay - methods
Liver - chemistry
Liver - immunology
Liver - ultrastructure
mitosis
Mitosis - physiology
Rats
SNARE
tether
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Summary: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.
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ISSN:1398-9219
1600-0854
DOI:10.1034/j.1600-0854.2003.00091.x