Proteomics/Protein Separations - Centrifugation/Density Gradient Centrifugation

Density Gradient Centrifugation
The use of density gradients has become almost routine in centrifugal fractionation of particle mixtures and purification of subcellular organelles and macromolecules. The basic idea behind the density gradient approach is that the mixture of particles to be separated is placed onto the surface of a vertical column of liquid, the density of which progressively increases from top to bottom, and then centrifuged. Although the particles in suspension are individually denser than the liquid at the top of the gradient, the average density for the sample (i.e. particles plus suspending liquid) is lower; only under such conditions could the sample zone be supported by the top of the density gradient. The two main types of density gradient centrifugation are rate-zonal separation and isopycnic separation.

Rate-Zonal Separation
In rate-zonal separation, particles are separated based on their size and mass. This means that they migrate through the gradient according to these properties - which allows their separation into distinct zones or bands, if they were layered as a thin zone onto the top of the gradient. This is useful in separating out particles with the same or very similar densities, but different sizes or masses: size is a much stronger discriminator than density in determining where particles migrate to in a given time, as sedimentation rate or velocity of a particle in a gravitational field is directly proportional to the density difference, but depends upon the square of the diameter.

Many proteins and other macromolecules, such as antibodies and virus particles, are isolated in this way.

It is generally the case that rate-zonal separations are dynamic rather than static: that is, if centrifugation is continued long enough, all the zones end up as one pellet at the bottom of the tube. It is also possible to combine rate zonal and density separations if the greatest density in the tube - for example, 1.04 to 1.23 g/cm3 for a 10 - 50% (w/v) sucrose gradient - is higher than one particular particle density, but less than the others being separated.

Isopycnic Separation
In isopycnic separation the particles migrate through the solvent gradient until they reach the point where their buoyant density is equal to that of the gradient. This is known as the isopycnic point or isodense position. Once the particles have reached their isopycnic point they will no longer move in the gradient, regardless of how much longer the centrifuge is run for. An example of this type of gradient is a caesium chloride concentration gradient. This is a salt that - for example - has a density of 1.08 g/cm3 for a 10% (w/v) solution, increasing to 1.58 g/cm3 for a 50% (w/v) solution. It and other heavy metal salts have the problems of being somewhat toxic and exerting a very strong osmotic pressure, as well as chemically affecting certain macromolecules. There are other, more inert materials better suited to isopycnic or even rate zonal separations, such as iodixanol: this is a non-ionic polymer which has very low osmolality and is capable of self-forming gradients like the heavy metal salts.

Rate vs. Isopycnic Separation: Advantages and Disadvantages
The choice of whether to try to isolate particles on rate basis or by isopycnic banding should take into account the following points:


 * For large particles such as cellular organelles, rate zonal runs are of considerably shorter duration than are isopycnic runs. This can be an important consideration where prolonged particle viability or liability are questionable.


 * Rate zonal runs are usually carried out in shallower density gradients than are isopycnic runs. Such condition can prove less damaging to particles that are osmotically active.


 * Rate zonal runs are usually carried out by using a lower RFC than is used in isopycnic runs. For certain especially fragile particles, the reduced hydrodynamic shear during sedimentation may be an aid in maintaining particle integrity.


 * The duration of centrifugation is not as critical in an isopycnic run as in a rate zonal run. To attain isopycnic banding, a certain minimum amount of centrifugation is required, but extending the centrifugation time beyond this does not much alter the results if the sample was layered on top of the gradient.


 * Generally speaking, greater numbers of particles can be separated by isopycnic density gradient centrifugation than by rate centrifugation. This is because the density gradients used for isopycnic runs are steeper and more stably support a greater quantity of sample.

Preparation of Density Gradients
Density Gradients are used not only for particle separations in the centrifuge tubes of conventional rotors, but also in special-purpose instruments such as zonal rotors, continuous flow rotors,and stay-put devices .The fundamentals of density gradient preparation are the same in nearly all the applications, but for the time being the discussion will center around gradient generation in tubes. Density Gradient may be subdivided into two main categories according to the means of preparation: step gradients and continuous gradients.

Step Gradients
Step Gradients are prepared by successively layering solutions of different density in the centrifuge tube and then layering the sample to be fractionated on top of the last "step". Step gradients are sometimes useful in density gradient centrifugation because the abrupt density steps that persist can be used as surfaces onto which particles can sediment during centrifugation. This results in discrete particle layers at each step. Special density gradient generating equipment is not required for production of step gradients. The gradient may be built up in the centrifuge tubes carefully layering one step on another, beginning with the densest step, by use of a pipette. Alternatively the gradient can be formed beginning with the least dense step by depositing each layer at the bottom of the centrifuge tube through a narrow cannula.

Continuous Gradients
Continuous density gradients are gradients in which the density changes smoothly and continuously from one limit or extreme to another. Such gradients can be produced from step gradients by allowing sufficient time for diffusion to smooth out the steps, but continuous gradients are normally prepared directly by using special devices known as gradient makers or gradient engines.

Gradient Materials
For most biological work, the ideal solute used to produce a density gradient should satisfy the following criteria:
 * The solute should be sufficiently soluble in aqueous media to produce the range of densities needed.
 * Dissolution of the solute should not result in solutions of high viscosity in the desired density range. Viscous solutions pose special mixing problems for gradient makers and are handled with some difficulty. Moreover, the sedimentation rate of a particle decreases directly in relation to the viscosity of the surrounding medium.
 * The solute should not exert too high an osmotic pressure in the desired density gradient range. The size, the shape, and the density of whole cells, membrane-enclosed subcellular organelles, and even macro molecules can be altered as these particles sediment through a density gradient if the osmotic pressure of the surrounding medium changes markedly.
 * Solutions of the gradient solute should be adjustable to the pH and ionic strengths that are compatible with the particles being separated.
 * The solute should not react with the particles being separated and should not interfere with the methods of analysis of the collected fractions.
 * Solutions of the gradient solute should exhibit a property that can conveniently be used as a measure of concentration or gradient density.
 * The solute should be readily removable from the collected gradient fractions (should that be necessary or desirable).
 * The solute should be affordable in the quantities needed.

Collecting Fractions
At the conclusion of centrifugation, the density gradient and the entangled particle zones are usually removed from the tube (or rotor) and collected as a series of fractions. This may be achieved in several ways. One of the oldest and simplest methods is to puncture the bottom of each centrifuge tube and allow the contents to slowly drip out. The gradient may also be removed by carefully lowering a narrow cannula to the bottom of the tube and withdrawing the gradient by use of a peristaltic pump. In both procedures the gradient exits the tube dense end first, and thus the separated particles are collected in order of decreasing sedimention rate or density. A third option is to puncture the tube at the location in the gradient of the material of interest and extract it manually. For a detailed description of this see the protocol at http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/23%20Plasmid%20Prep.pdf. The diagram below demonstrates this process in the collection of the plasmid DNA fraction; however, one can see how easily this could be applied to the protein fraction instead.



Measuring Gradient Density
Since the density of a solution is its mass:volume ratio, measurement of these two parameters in successive aliquots of the collected gradient reveals the gradient's profile. However this approach is awkward and time consuming and is rarely used in connection with density gradient centrifugation. Instead, the refractive index of small drops of liquid taken from different regions of the gradient is determined, and the corresponding densities obtained from tables or curves relating refractive index to density.

Light Refraction and Snell's Law
The speed of the light changes as light passes from one medium into another. If the light passes across the interface between the two media at any angle other than 90 degree, the direction of the light is also changed. The refractive index of a substance is a measure of its ability to bend the light and is related to the substance's density. For more detailed information on this subject see Snell's law at Wikipedia.

Next Section: Differential Centrifugation