Isolation of golgi transmembrane protein - (Feb/06/2008 )

I was hoping for a protocol that is good for differential density centrifugation so that I may just isolate the golgi. Thanks

What about 1000g for 10 min - pellets nucleus
Take supernatent spin for 10,000g for 30 min - pellets the mitochondria
Then the remaining supernatent should have Golgi, ER, other membrane fragments and ribosomes


After that I am unsure about isolating Golgi

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the supernatant after the 10,000xg run it on a sucrose gradient or any other density gradient (ficoll, iodaxanol) and check each fraction from the gradient with golgi markers to see where the peak.

Instead of 10,000 x g, do a 14,000xg, as you might still get some mitochondria at 14,000x g

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One protocol states:

Golgi apparatus was isolated by rate zonal and isopycnic centrifugation of a cauliflower homogenate according to [14]. The homogenate was centrifuged at 6000×g for 15 min and the supernatant was layered over a 50% and 45.4% (w/w) sucrose step gradient and centrifuged at 30 000×g for 30 min. The supernatant above the particulate material at the 45.4% sucrose interface was removed and aliquots of 43%, 37.5% and 17% w/w sucrose solution were carefully layered on top of the particulate material and centrifuged at 100 000×g for 3 h. The 17/37.5% and the 37.5/43% sucrose interfaces are enriched in membranes of the Golgi apparatus. Each interface was collected, diluted with 0.2 M sucrose solution, centrifuged at 100 000×g for 30 min and resuspended in basic buffer. All sucrose solutions were made in basic buffer.

When it says that "interfaces are enriched in membranes in the Golgi apparatus does that mean that the Golgi apparatus will not be intact. Being intact would be ideal.

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Interface is where you will find the membranes. Now the cells are going through a lot of manipulation and this can lead to a percentage of the golgi being damaged. There will be lots of smaller golgi vesicles which will be intact.

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isolation is impossible but high enrichment in sucrose gradient and sharp fractionation should succeed

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