Endocytosis analysis – a review of density gradient methods

Some of the first reports on the use of Nycodenz® for the fractionation of endosomes were published in the mid-nineteen eighties by Howard Evans and his co-workers, working at the National Institute for Medical Research in London. 

They worked primarily on the endocytosis of a variety of 125I-labelled asialo-glycoproteins by the perfused rat liver. After differential centrifugation of the homogenate, a supernatant from a light mitochondrial pellet was first applied to a continuous 15-43% (w/v) sucrose gradient (over layers of 43% and 70% sucrose) and centrifuged at 140,000 g for 3.5 h.

The lightest fraction (1.095-1.117 g/ml) was re-centrifuged on a 13.8-27.6% (w/v) Nycodenz® gradient at 110,000 g for 18 h. Two very distinct, well-separated peaks of radioactivity were obtained at 1.090 and 1.115 g/ml. The denser material was identified as early endosomes and the lighter as late endosomes