The four protein purification techniques are discussed in detail in this article.
These are the four strategies for protein purification:
(2) Differential Solubilization and Precipitation
(3) The use of ultracentrifugation
Analytical and preparative methods can be used for protein purification, respectively.
The amount of protein that can practically be purified using that method serves as the deciding factor, though the distinction is not precise. While preparative methods aim to make large quantities of the protein for other uses, like structural biology or industrial use, analytical methods aim to find and identify a protein in a mixture. In general, analytical applications can use preparative methods but not the other way around.
Method #1: Depending on the source,
the protein must be dissolved by rupturing the tissue or cells that contain it. The process of repeatedly freezing and thawing, sonication, homogenization under high pressure, or permeabilization using organic solvents are a few ways to accomplish this. Depending on how delicate the protein is and how robust the cells are, a particular method is best.
Centrifugation can be used to separate soluble protein from cell membranes, DNA, etc. after the extraction process. Proteases, which will begin breaking down the proteins in the solution, are also extracted during the extraction process. It is typically preferable to move quickly and keep the extract cool to slow down proteolysis if the protein is proteolysis-sensitive.
Method #2:Precipitation and Differential Solubilization,
Precipitation with ammonium sulphate (NH4)2SO4 is a typical first step in bulk protein purification to isolate proteins. This is accomplished by varying the ammonium sulphate addition rates while gathering the various precipitate protein fractions. This method has the benefit of being able to handle very large volumes at a low cost.
Water-soluble proteins are the first to be purified. To isolate any one specific protein from others that are in the same membrane compartment during the purification of integral membrane proteins, the cell membrane must be disrupted. Sometimes it is possible to isolate a specific membrane traction first, for example, isolating mitochondria from cells prior to purifying a protein found in a mitochondrial membrane.
To dissolve cell membranes and keep membrane proteins in solution during purification, a detergent like sodium dodecyl sulphate (SDS) can be used; however, because SDS causes denaturation, milder detergents like Triton X-100 or CHAPS can be used to maintain the protein’s native conformation throughout purification.
Method #3: is called ultracentrifugation,
and it involves using centrifugal force to separate mixtures of particles suspended in liquid that have different masses or densities. High-speed rotation of a container (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, results in an outward force that is proportional to each particle’s mass.
The resistance the liquid imposes on the particle balances the tendency of a given particle to move through the liquid as a result of this force. Massive, small, and dense particles move outward more quickly than less massive particles or particles with more “drag” in the liquid as a result of the sample being “spun” in a centrifuge.
When particle suspensions are “spun” in a centrifuge, a “pellet” that is enriched for the most massive particles with little drag in the liquid may form at the bottom of the vessel. The “supernatant” is the term for the remaining, non-compacted particles that are primarily still in the liquid and can be removed from the vessel to be separated from the pellet.
The sample’s applied angular acceleration, which is typically measured in relation to the g, determines the rate of centrifugation. If samples are centrifuged for a sufficient amount of time, the particles in the vessel will reach equilibrium, accumulating specifically at a point where the buoyant density of the particles balances the centrifugal force. Such “equilibrium” centrifugation enables thorough particle purification.
Sucrose gradient centrifugation:
A tube is used to create a linear gradient of sugar concentration, usually sucrose glycerol or Percoll, with the highest concentration at the bottom and lowest concentration at the top. The gradient is then covered with a protein sample, which is then spun rapidly in an ultracentrifuge. As a result, heavier macromolecules migrate to the tube’s bottom more quickly than lighter molecules.
Particles experience increasing centrifugal force during centrifugation in the absence of sucrose as they move away from the centre of rotation (the further they move, the faster they move). The issue with this is that there is only a small observable window for the useful separation range inside the vessel.
The particle of interest will move considerably further when a sample is spun twice as long, not twice as far. However, as the proteins pass through a gradient of sucrose, they come into contact with a liquid that is getting denser and more viscous.
The centrifugal force will be balanced by a properly constructed sucrose gradient, causing the particles to move roughly in proportion to the amount of time they have spent in the field. Rate zonal centrifugations are used to separate samples using these gradients. The gradient is then fractionated and collected after the protein/particles have been separated.
Method #4: chromatographic methods,
which typically include one or more steps in a protein purification protocol. The fundamental step in chromatography is the flow of the protein-containing solution through a column that is filled with a variety of materials. The time needed to pass the column or the conditions needed to elute the protein from the column can be used to distinguish between different proteins because they interact differently with the material of the column. Typically, proteins are identified as they exit the column by their 280 nm absorbance.