Although analysing protein complexes is just as important, analysing individual ones is fundamental. When it comes to studying individual proteins, protein purification is king. Various resins are used as a part of the protein purification process.
There are four main methods of protein purification – extraction, precipitation and differential solubilisation, ultracentrifugation, and chromatographic methods. In this post, you’ll learn more about each one.
To bring the protein into the solution, you have to break the cell tissue where the protein is contained. The process of extraction is done using several different methods: repeated freezing/thawing, homogenisation using high pressure, sonication, and permeabilization using organic solvents. Choose between these methods based on the protein fragility and the cell sturdiness.
After selecting the right method and finishing the extraction process, the protein is located in the solvent. Now, using the process of centrifugation, the protein is separated from the DNA, or from the cell membranes.
Keep in mind that during the process of extraction, proteases are extracted, as well. Proteases will start digesting proteins located inside the solution. Depending on how proteolysis-sensitive the protein in question is, you should proceed quickly, to avoid protein damage. The extract is also usually kept cooled, which slows down the proteolysis process.
Precipitation and Differential Solubilisation
Precipitation uses ammonium sulphate to isolate proteins. This is a vital step in bulk protein purification. The process involves increasing the ammonium sulphate amounts gradually and then collecting the precipitate protein fractions. One of the main advantages of precipitation is that it can be used in large volumes while remaining inexpensive.
During this process, the first proteins that are purified are those that are water-soluble. To purify integral membrane proteins, cell membrane disruption is required.
A detergent dissolves the cell membranes. Sodium dodecyl sulphate, or SDS, is an option here. SDS, however, causes denaturation at unacceptable levels for some proteins. This is why milder detergents, like CHAPS or Triton X-100are, often applied.
To separate particle mixtures of varying densities and masses, the process of centrifugation is applied. Let’s say that you’re using a tube to contain your target protein mixture. When rotated at high speeds, thanks to the angular momentum-delivered outward force, the heavier particles are drawn outwards. The massive, dense, and small particles will move outward faster than lighter particles.
A pellet may form at the bottom of the tube that’s rich with the largest particles in the solution.
The centrifugation rate is measured compared to the G-force. Centrifuge the samples for long enough, and the particles within the vessel will eventually reach an equilibrium. This equilibrium is reflected in the particles accumulating specifically at the part of the tube where the density is balanced with the centrifugal force. This allows for advanced and extensive purification.
Ultracentrifugation is based on extensive centrifugation, which leads to advanced protein purification potential.
Almost every protein purification protocol includes at least one chromatographic step. In most cases, a combination of chromatographic steps is used. The procedure here is based on flowing a solution through columns that contain various materials.
Most proteins act specifically with various column material. So, each protein has different ideal chromatography method combinations. Here are a few types of chromatographic methods to keep in mind.
Size Exclusion Chromatography (SEC)
SEC utilises porous gels to separate the protein inside the solution. This entire method is based on the fact that smaller molecules have more volume to cover when moving through the matrix.
Ion Exchange Chromatography (IEC)
This method is based on separating the compounds, based on the degree and nature of their ionic charge.
Affinity Chromatography (AC)
This separation technique is based on molecular conformation. In this method, various specific resins are used.
This technique is based on making a sequence of six to eight histidines, constructing the protein’s C-terminal. The polyhistidine-tag (an amino acid in proteins) will bind to divalent metal ions (nickel and cobalt, for instance).
Other chromatographic methods can also be used in combination with those outlined to achieve the desired levels of protein purification.
Protein Purification Methods
Depending on what your needs are, you can go with different types of purification, even, as mentioned, use the methods in combination. Experimentation will lead you to find your ideal proteinpurification method for each protein type that you’re using.