In biochemistry, a protein is a polymer of amino acids arranged in the form of polypeptide chains. Proteins play an integral role in cell structure and function.
In order to study proteins, however, it is important to first identify and isolate them. To accomplish this, biochemists carry out what’s regularly known as high-throughput protein purification. First, individual proteins are isolated, then they are separated based on molecular weight, solubility, charge, and specific binding affinity. This process helps researchers analyze individual proteins and study protein characterization and interactions.
Protein purification can be defined as a series of steps that are carried out in order to obtain and study the desired protein from a complex mixture. Isolating a protein will help in the analysis of the protein’s:
This process holds many experimental applications, especially in the large-scale production of recombinant proteins. (However, purification of native proteins is often challenging. In such cases, affinity tags like Polyhistidine, Glutathione-S-transferase, and His-tags are used to isolate and immobilize them.)
In this article, we provide an overview of the purification steps, methods, and strategies involved.
To successfully isolate a protein, it must undergo the process of protein purification. The general steps of the process are outlined below:
Cell lysis is the disruption of cells and denaturing of proteins using various enzymatic methods (protease inhibitors), chemical methods (detergents), or physical methods (sonication). As a result, the cell lysate will contain both soluble and insoluble proteins, nucleic acids, cell debris, organelles, and membrane remains.
The goal, however, is to obtain only the proteins that are to be purified. To do so, cells undergo the next step of clarification to remove all the contaminants. This is achieved by filtration or centrifugation.
Once achieved, the protein extract is ready for concentration.
The next step is to isolate the protein from the extract. This is also sometimes called the recovery stage. Here, the protein is isolated by binding the protein molecule to a matrix; this is carried out with techniques like affinity chromatography and magnetic bead separation.
Elution is the process of extracting one material that is adsorbed to another by washing with a solvent. An appropriate elution buffer is used to wash away all the non-specific binding on the column. The number and length of washes will depend on the type of column used.
Elution of the desired protein is performed by changing the pH of the column; the charged functional groups of proteins neutralize, and they can thus be extracted. The process involves the use of an elution buffer and reagents like imidazole (used in His-tagged proteins), which effectively elute the target protein in high concentrations. This process helps researchers obtain high protein concentrations for further analysis.
There are four main different methods for carrying out the purification of proteins. Let’s review each in more detail below:
Chromatography is a common technique used to obtain the target protein sample. It separates substances according to the difference in their properties. It is a popularly known purification technique and involves the use of columns filled with resin, from which the protein of interest is to be extracted.
Chromatography techniques work on the principle of a stationary phase and a mobile phase, where the stationary phase is the resin in the column, and the mobile phase is the liquid solvent. The protein of interest (or the impurities in some cases) binds to the ligand in the stationary phase, while each mobile phase either removes impurities or elutes the protein.
A common chromatography technique is high-performance liquid chromatography (HPLC). Other types of chromatography techniques can be employed based on the protein to be purified, such as:
Isolation is the simplest of all protein purification strategies as the pure protein, in this case, is generally obtained by means of:
Another purification method is salt precipitation. Salt precipitation is not as popular or commonly used today because it doesn’t always yield highly purified proteins.
This purification process effectively “salt outs” or culls protein from the mixture. Precipitation is carried out by introducing high osmolarity, using ammonium sulfate as the salt.
Different proteins precipitate at different concentrations of ammonium sulfate. Once the protein is precipitated, the remaining salts in the supernatant are removed by dialysis through filtration or gel exclusion chromatography.
Immunoblotting is a highly used technique to visualize proteins and is sometimes carried out in combination with affinity chromatography. It is a rapid and sensitive assay majorly employed for the detection of proteins.
Immunoblotting for proteins is also known as western blotting. In a typical western blot, antibodies are used to detect the desired protein.
The proteins are first separated by their size using SDS-PAGE (sodium dodecyl sulfate gel electrophoresis). They are then transferred to a porous membrane and blocked to prevent any non-specific binding of antibodies.
After blocking, the membrane is incubated with a primary antibody specific to the protein of interest. Then, it is incubated with a secondary antibody specific to the primary antibody. The secondary antibody is usually conjugated with an enzyme that produces a signal for detection. Thus, only the protein of interest will be signaled. Such protein extraction methods also help indicate protein expression.
Don’t have the budget to purchase lab equipment outright? Consider leasing through Excedr to save your lab time and money. Browse your leasing options today!
Protein purification has now become a basic requirement in biomedical research. With an increased number of proteins being discovered, it has become important to analyze their structure, function, and interactions with other biomolecules.
To carry out the purification methods with accuracy, labs need high-quality chemicals and equipment.
Contact us to learn more today.