Density gradient centrifugation
Gradient stability
Density gradients have unlimited potential for separation of biomolecules. Gradient stability as well as its capacity for separation are important. The top of the gradient must be dense enough to support the sample and the column must have enough of a density gradient to remain stable during the centrifugation and fractionation of the tube. Thus, a 15-20% sucrose gradient will be much less stable than a 5-45% gradient. The greater the change in % between the top and bottom of a gradients, the more stable it is. As a general rule, the delta % should be ≥20% and as high as 40%.
The choice of solute; density, viscosity, osmolarity
The choice of solute is dictated by the type of separation desired:
Sucrose: The most common solute. It is cheap, very soluble, chemically inert and does not absorb in the UV. However, concentrated solutions of sucrose have high osmotic potential that cause shrinkage of certain cells and organelles.
Glycerol: A substitute for sucrose, sharing most of its properties. However, the high viscosity of glycerol reduces the effective density range and glycerol appears to inhibit some enzyme activities.
CsCl (NaCl, KCl, KBr, LiCl): These salt gradients are useful when nucleic acids are banded, since the high solubility and density of some of these solutes can float these dense biomolecules. These gradients lack the viscosity of sucrose gradients and are less stable as a result.
K Acetate: Used in velocity banding of DNA/RNA fragments which are recovered from the fractions by adding isopropanol and precipitating the nucleic acid.
Ficoll or Dextran: A high molecular weight polymer of sucrose or glucose used in some cell separations
Fig. 1. 26S proteasome isolated from yeast Saccharomyces Cerevisiae and fractionated on 15-35% sucrose gradients
Fig. 2 . Phycobilisome bands from Chondrus crispus isolated on sucrose gradients (1)
1. M.L. Cornish, S.J.B. O'Leary, D.J. Garbary (2013) Phycobilisome composition in Chondrus crispus (Gigartinales, Rhodophyta) from a wild type strain and its vegetatively derived green mutant, Algae, 28, 121-129