I want to isolate Golgi vesicles from Hela cells by flotation through a discontinuous sucrose gradient. Theoretically, Golgi band will be harvested from the 0.8 M/1.2 M sucrose interface.
But I found nothing near the 0.8 M/1.2 M sucrose interface. However, after Centrifuging 2 h in an ultracentrifuge at 110,000 × g, 4°C, an obvious band was found from the 1.4 M/1.6 M sucrose interface. Golgi markers (GM130, TGN46), ER marker (CALR), mitochondria marker (COX4) and nucleus marker (histone H3) were detected by western blot.
Is there any experience about isolating the Golgi from cultured cells?