Biochemical Techniques and Instrumentation

4.1.5. Density gradient centrifugation

Density gradient centrifugation is the preferred method to purify subcellular organelles and macromolecules. Density gradients are generated by placing layer after layer of gradient media such as sucrose or caesium chloride (CsCl) in a tube with the heaviest layer at the bottom and the lightest at the top either in a discontinuous or continuous mode. Cell fraction to be separated is placed on top of the layer, centrifuged and separated according to their size or densities. The density gradient separation is classified further into two categories: rate-zonal (size) separation and isopycnic (density) separation.

1. Rate zonal (size) separation

Rate-zonal separation depends on particle size and mass instead of particle density for sedimentation. Most common applications include separation of cellular organelles such as endosomes or antibodies. Antibody classes generally have very similar densities but different masses; and can be separated based on density. 

2. Isopycnic (density) separation

Isopycnic separation depends on particle density. A particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is achieved, the length of centrifugation does not have any influence on the migration of the particle. Most common application is separation of nucleic acids in a caesium chloride (CsCl) gradient and cell organelles in a surcose gradient. A variety of gradient media can be used for isopycnic separations (Table 2).

Table 2. Applications of density gradient media for isopycnic separations

Gradient	Cells	Viruses	Organelles	Nucleoproteins	Macromolecules
Sugar (sucrose)	+	+++	+++	+	-
Polysaccharides (Ficoll)	++	++	++	-	-
Colloidal silica (Percoll)	+++	+	+++	-	-
Iodinated media (Nycodenz)	++++	++	++++	+++	+
Alkaline metal salts (CsCl)	-	++	-	++	++++

++++ excellent; +++ good; ++ good for some applications; + limited use; - unsatisfactory