Methods of protein purification

The methods used in protein purification, can roughly be divided into analytical and preparative methods. The distinction is not exact, but the deciding factor is the amount of protein, that can practically be purified with that method. Analytical methods aim to detect and identify a protein in a mixture, where as preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use. In general, the preparative methods can be used in analytical applications, but not the other way around.

Extraction
Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it. There are several methods to achieve this: Repeated freezing and thawing, sonication, homogenization by high pressure or permeabilization by organic solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are. After this extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation. The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.

Precipitation and differential solubilization
In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. One advantage of this method is that it can be performed inexpensively with very large volumes.

The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein's native conformation during complete purification.

Ultracentrifugation
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the angular momentum yields an outward force to each particle that is proportional to its mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of "spinning" the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more "drag" in the liquid. When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid. The remaining, non-compacted particles still remaining mostly in the liquid are called the "supernatant" and can be removed from the vessel to separate the supernatant from the pellet. The rate of centrifugation is specified by the angular acceleration applied to the sample, typically measured in comparison to the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium" centrifugation can allow extensive purification of a given particle.

Sucrose gradient centrifugation — a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica based density gradient media, like Percoll) is generated in a tube such that the highest concentration is on the bottom and lowest on top. Percoll is a trademark owned by GE Healthcare companies. A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. During centrifugation in the absence of sucrose, as particles move farther and farther from the center of rotation, they experience more and more centrifugal force (the further they move, the faster they move). The problem with this is that the useful separation range of within the vessel is restricted to a small observable window. Spinning a sample twice as long doesn't mean the particle of interest will go twice as far, in fact, it will go significantly further. However, when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity. A properly designed sucrose gradient will counteract the increasing centrifugal force so the particles move in close proportion to the time they have been in the centrifugal field. Samples separated by these gradients are referred to as "rate zonal" centrifugations. After separating the protein/particles, the gradient is then fractionated and collected.

Chromatographic methods
Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:

Size exclusion chromatography
Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluant (solvent) before being collected at the other end of the column of gel.

In the context of protein purification, the eluant is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.