Protein purification involves isolating a specific protein from a complex mixture of biological materials. This process separates the desired protein from contaminants. The goal is to obtain the protein in a pure form for detailed study of its structure, function, and interactions.
Why Proteins Are Isolated
Scientists and industries isolate pure proteins for various reasons. Purifying a protein allows researchers to study its biochemical properties, such as enzyme activity or binding affinity, providing insights into biological processes and disease mechanisms.
Pure proteins are also widely used in medicine, particularly for developing therapeutics. Many drugs, like insulin or monoclonal antibodies, are protein-based. Ensuring their high purity is important for safety and effectiveness, as impurities could lead to adverse effects or reduced efficacy. Beyond medicine, purified proteins, such as enzymes, find uses in industrial applications like detergents and food processing and in diagnostic tools for detecting disease markers.
General Stages of Protein Purification
Protein purification typically follows a series of steps to isolate the target protein. Initial preparation involves the biological material, often by breaking open cells or tissues to release their contents, a process known as cell lysis.
Following cell lysis, a separation step removes large cellular debris. Centrifugation achieves this by spinning the sample at high speeds, forcing heavier components to settle while proteins remain in the liquid portion. The supernatant may then be concentrated to reduce its volume. The final stages involve targeted purification techniques that separate the specific protein from other remaining proteins and small molecules.
Common Isolation Techniques
Protein purification relies on various techniques that exploit differences in protein properties like size, charge, and binding affinity. Chromatography is a widely used method for separating proteins. It involves passing a protein mixture through a stationary phase, where proteins interact differently.
Ion Exchange Chromatography
Ion exchange chromatography separates proteins based on their net electrical charge. Proteins bind reversibly to a charged resin in a column. They are then eluted by gradually changing the salt concentration or pH of the buffer.
Size Exclusion Chromatography
Size exclusion chromatography (also called gel filtration) separates proteins based on their molecular size. Larger proteins pass through porous beads in the column more quickly. Smaller proteins enter the pores and are retained longer, eluting later.
Affinity Chromatography
Affinity chromatography is highly specific, exploiting a protein’s ability to bind to a particular molecule (ligand) immobilized on a resin. The target protein binds to the ligand, while other proteins wash through. Isolation occurs by disrupting this specific binding.
Electrophoresis (SDS-PAGE)
Electrophoresis, particularly SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), is a common technique for separating proteins by their molecular weight. Proteins are denatured and coated with a negative charge by SDS, causing them to migrate through a polyacrylamide gel matrix when an electric field is applied. Smaller proteins move faster through the gel, creating distinct, visible bands. While SDS-PAGE is excellent for analyzing purity and estimating molecular weight, it generally denatures proteins and is not typically used for purifying large quantities for functional studies.
Other Separation Methods
Other methods like filtration and dialysis are used for buffer exchange or removing small molecules like salts. Dialysis involves placing the protein sample in a semi-permeable membrane, allowing small molecules to diffuse out while retaining the larger proteins.
Assessing Purity and Activity
After purification, scientists must confirm that the isolated protein is pure and remains functional. Electrophoresis, such as SDS-PAGE, is frequently used to visualize proteins in a sample; a single band on the gel suggests high purity. Spectrophotometric methods like UV-Vis and Bradford assays can quantify total protein concentration, but activity assays are more specific as they measure only the active protein.
Beyond purity, it is important to assess the protein’s activity or functionality to ensure expected function. Enzyme assays measure catalytic activity, while binding studies are performed for proteins that bind to other molecules. This step verifies that the protein’s biological function has been preserved. Researchers also consider the yield, the amount of purified protein obtained, as sufficient quantities are important for downstream applications.