Protein is the main substance of human tissues and organs and participates in various forms of life activities. Protein separation and purification is the crucial premise for studying the structure and function of various proteins. The methods for protein purification range from simple precipitation procedures to sophisticated chromatographic and affinity techniques. Generally, a single separation technology cannot achieve the desired purification effect, which requires the combination of multiple technologies. Crude separation technologies of the target protein are salting out, isoelectric precipitation, organic solvent precipitation, dialysis, and ultrafiltration with disadvantages such as low resolution and plenty of impurities. Salting out is low cost, environmentally friendly and does not cause protein denaturation. On the contrary, although organic solvent precipitation has high separation ability, it can easily cause denaturation and inactivation of bioactive proteins. Both need to be combined with subsequent dialysis or ultrafiltration to remove salts or organic solvents. Because the isoelectric points of many proteins are very close, the separation ability of isoelectric precipitation is low and tends to be combined other methods to improve the separation effect.
Chromatography, electrophoresis, and molecular imprinting belong to high-precision separation techniques that can be used to further purify the desired protein. High performance liquid chromatography (HPLC) is a widely used technology in laboratory protein separation with the advantages of high efficiency, rapidity, and easy automation. Gel filtration chromatography (also called steric exclusion chromatography) is a method that uses differences in molecular weight or shape of proteins for separation. Gel filtration chromatography usually deals only with natural proteins, while electrophoresis usually involves separating and denaturing polypeptides. Electrophoresis and ion-exchange chromatography exploit the overall charge of proteins to purity them. Affinity techniques are used for separating the desired protein from others by a biospecific property of the protein. Among them, immunoaffinity chromatography is the most specific and effective protein purification technique. It exploits the principle of biomolecular recognition, that is, the ability of bioactive macromolecules to form specific and reversible complexes with affinity ligands. In the affinity chromatography, immobilization of the ligand to which the protein binds (or of antibody to the protein) enables selective adsorption of the desired protein. Affinity ligands are either biological ligands or synthetic ligands. Biological ligands have high affinity and binding capacity, while synthetic ligands generally show higher chemical and biological stability and higher cost-effective.
Prior to embarking upon the purification of a protein, the general considerations to be undertaken in designing the purification are biochemical characteristics of the protein of interest, the downstream application, and the required amount of purified protein. Other considerations related to the purification process itself are sensitivity, accuracy, precision, linearity, substrate availability, and cost. Amerigo Scientific offers a wide range of protein purification products including various reagents, columns, kits, and related products designed for high-yield purification of proteins.