Two-dimensional gel electrophoresis is a laboratory technique used to separate a complex mixture of proteins from a biological sample. This method resolves individual proteins from a mixture containing potentially thousands of different types. The separation is accomplished by targeting two distinct and independent physical properties of the proteins.
The Two-Dimensional Separation Process
The first step is isoelectric focusing (IEF), where proteins are separated based on their isoelectric point (pI). The isoelectric point is the specific pH at which a protein has no net electrical charge. The protein sample is loaded onto a gel strip with an immobilized pH gradient. When an electric field is applied, proteins migrate through the gradient until they reach the pH that matches their pI, at which point they become neutral and stop moving.
After the separation by charge, the gel strip is subjected to a second separation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Before this step, the strip is treated with the detergent sodium dodecyl sulfate (SDS). This chemical denatures the proteins, unfolding them into linear chains and coating them with a uniform negative charge.
With their native charge eliminated, the proteins now carry a negative charge proportional to their length. A second electric field is applied at a 90-degree angle, causing the proteins to migrate into a new gel. In this dimension, their movement is determined by their molecular weight, or size, with smaller proteins moving faster and further than larger ones.
Visualizing and Interpreting the Results
The result is a gel displaying a two-dimensional map where each spot ideally corresponds to a single protein. The position of each spot reveals the protein’s physical properties. The horizontal axis represents the pH gradient from isoelectric focusing, indicating the protein’s isoelectric point. The vertical axis represents separation by size from SDS-PAGE, indicating the protein’s molecular weight.
To make the separated protein spots visible, the gel must be treated with a stain. The choice of stain depends on the required sensitivity and the amount of protein present. Coomassie Brilliant Blue is a common stain that binds to proteins, rendering them as visible blue spots against a clear background. For detecting smaller quantities of protein, more sensitive methods like silver staining are used, which can reveal spots containing only nanogram amounts.
Key Applications in Scientific Research
This detailed protein mapping has applications in proteomics, the large-scale study of proteins. A primary use is in biomarker discovery. By comparing protein maps from healthy and diseased tissues, researchers can identify proteins that are more or less abundant. These differentially expressed proteins can serve as potential biomarkers for diagnosing diseases or monitoring treatment effectiveness.
The technique also helps in understanding cellular processes. Scientists can observe how the protein landscape of a cell changes when exposed to conditions like a new drug, a toxin, or an environmental stressor. These changes in protein expression provide insights into the molecular mechanisms cells use to respond and adapt.
Beyond basic research, 2D electrophoresis is used in various industries. The biopharmaceutical field uses it for quality control to ensure the purity and consistency of protein-based drugs, verifying the correct protein is present and free from contaminants. The food science industry uses it to analyze food protein composition for allergy research and product authentication.
Technical Considerations and Complementary Methods
The execution of 2D electrophoresis is a labor-intensive process. Factors during sample preparation, like ensuring proteins are properly solubilized, can impact the final outcome. The technique may also face challenges in separating certain classes of proteins, such as those that are very large, very small, or embedded within cell membranes.
Due to these factors, 2D electrophoresis is often one step in a larger analytical workflow. While the gel provides a map of protein quantity and physical properties, it does not identify them. To achieve this, researchers use the complementary technique of mass spectrometry. A protein spot is physically cut from the gel and analyzed by a mass spectrometer, which determines its precise identity by measuring the mass of its constituent fragments.