What Are the Main Methods for Protein Separation and Purification and Their Underlying Principles?
Below are some commonly employed methods for protein separation and purification, along with their fundamental principles:
Salting Out
This method involves gradually increasing the salt concentration (e.g., ammonium chloride) to induce protein precipitation. Since the solubility of proteins varies depending on salt concentration, selective precipitation enables partial purification.
Electrophoresis
Proteins are separated based on their mobility in an electric field. Common electrophoretic techniques include one-dimensional and two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF).
Liquid Chromatography
Protein separation is achieved based on differential interactions with chromatographic stationary phases. Major liquid chromatography techniques include:
1. Affinity Chromatography
Exploits the highly specific binding affinity between a protein and its corresponding ligand to facilitate separation.
2. Ion Exchange Chromatography
Separates proteins based on electrostatic interactions between their surface charges and ion exchange resins.
3. Size Exclusion Chromatography (SEC)
Fractionates proteins according to their molecular size, as smaller molecules penetrate the gel matrix more deeply than larger ones.
4. Hydrophobic Interaction Chromatography (HIC)
Utilizes hydrophobic interactions between proteins and hydrophobic resins to achieve separation.
Ultrafiltration
Employs semipermeable membranes with defined molecular weight cut-offs (MWCO) to separate proteins based on size. Large proteins are retained on one side of the membrane, whereas smaller molecules diffuse into the filtrate.
Protein Precipitation
Induces protein precipitation by altering physicochemical conditions such as temperature, solvent concentration (e.g., high alcohol content), or pH. The precipitated proteins are subsequently collected by centrifugation.
These methods may be used individually or in combination to achieve higher protein purity. The selection of an appropriate method depends on the physicochemical properties of the target protein, its source, and the specific research objectives.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
