Analysis of ER, plasma membrane, endosomes, Golgi, ERGIC and TGN from mammalian cells and tissues in continuous gradients (1.5-3 h spin)

The protocol described in this Application Sheet is based on methods first published by Yang et al [1] and Zhang et al [2]. Yang et al [1] used a linear 0-26% (w/v) iodixanol gradient to study the localization of UBC6 ubiquitin-containing protein in COS-7 cells. 

By using the gradient to separate endoplasmic reticulum (ER) and Golgi, they established that the transmembrane domain of a carboxylterminal anchored protein predisposes it to locate to the ER, while modulation of this domain resulted in re-targeting of the protein to the Golgi. 

Zhang et al [2] used a 1-20% (w/v) iodixanol gradient also to separate the ER and Golgi from transfected CHO and human embryonic kidney (HEK293) cells. The authors showed that the full-length presenilins (PS1 and PS2) were located in the ER while N- and Cterminal fragments were distributed to the Golgi membranes.