Analysis of membrane trafficking in mammalian tissues and cells: fractionation of ER, Golgi, TGN, PM and endosomes

One of the first published papers to report the use of iodixanol gradients for the analysis of endoplasmic reticulum (ER), Golgi and plasma membrane (PM) from COS-7 cells was by Yang et al [1] in 1997. 

A post-nuclear supernatant, loaded on to a 0-26% (w/v) iodixanol gradient was centrifuged at 200,000 g for approx. 2 h. Analysis of the gradient established that the density of the three major membrane compartments increased in the order PM

Some workers maintain that the highest resolution of membrane vesicles is only obtained if the centrifugation is carried out at relatives low RCFs for extended time periods; for example 50,000-100,000 g for 18 h. 

An example is given in Figure 2; an 8-34% (w/v) iodixanol gradient was used to study the processing of the amyloid β peptide From CHO cells stably transfected with PS1 [2]. 

The gradient gave excellent resolution of early endosomes Golgi and ER. The method has also been able to resolve perinuclear ER from the bulk ER of mouse 3T3 fibroblasts [3]. This long-spin strategy however is not the only one that provides the ability to fractionate sub-domains of the same membrane compartment (see Section 2)