Gel electrophoresis is a laboratory technique used to separate and analyze large biological molecules, such as DNA, RNA, and proteins. Its primary purpose is to distinguish these molecules by properties like size, charge, and shape, making it a fundamental tool in molecular biology.
The Electric Force
The fundamental principle driving gel electrophoresis is the application of an electric field across a gel matrix. An electrophoresis apparatus consists of a gel placed within a chamber filled with a buffer solution, connected to a power supply with a negative electrode (cathode) at one end and a positive electrode (anode) at the other. The activated power supply generates an electric current within the gel.
Biological macromolecules typically carry a net electrical charge. For instance, DNA and RNA molecules are inherently negatively charged due to the phosphate groups in their backbones. In contrast, proteins can exhibit positive, negative, or neutral charges depending on their amino acid composition and the pH of the surrounding buffer. This charge causes molecules to migrate through the gel towards the oppositely charged pole. Negatively charged nucleic acids, for example, move towards the positive electrode, while positively charged proteins move towards the negative electrode.
The Gel Matrix
The gel itself serves as a porous matrix, functioning like a molecular sieve through which charged molecules navigate. Common gel materials include agarose, typically used for separating larger molecules like DNA and RNA, and polyacrylamide, preferred for proteins and smaller nucleic acids due to its finer resolution capabilities. These gels are formed by a network of interconnected molecules, creating microscopic pores.
The size and density of these pores are controllable by adjusting the concentration of the gel material. For example, a higher concentration of agarose results in smaller pores, which improves the separation of smaller DNA fragments, while lower concentrations allow for the resolution of larger fragments. Similarly, increasing the acrylamide concentration in polyacrylamide gels decreases pore size. As molecules move through this porous structure, the gel matrix provides resistance, impeding progress. Larger molecules encounter greater resistance and move more slowly, while smaller molecules navigate the pores with less impedance.
Molecular Properties and Separation
Separation in gel electrophoresis combines the electric force and the gel’s sieving effect, influenced by molecular properties. The net charge of a molecule plays a role, as molecules with a greater net charge generally experience a stronger electric force and thus migrate faster. However, for DNA and RNA, their charge-to-mass ratio is relatively constant because each nucleotide carries a similar negative charge. Consequently, for nucleic acids, migration speed is primarily determined by their size, with smaller fragments moving more rapidly than larger ones.
Molecular size is a dominant factor. Smaller molecules pass through the gel’s pores more easily and quickly, whereas larger molecules are significantly hindered by the gel’s matrix, causing them to move more slowly. This differential migration based on size results in distinct bands forming along the gel, with smaller molecules traveling further. Beyond size and charge, the three-dimensional shape or conformation of a molecule can also impact its migration rate. For example, in DNA, supercoiled forms tend to be more compact and typically migrate faster than linear or relaxed circular forms of the same molecular weight. For proteins, which have diverse shapes and charges, they are often treated with a detergent like sodium dodecyl sulfate (SDS) to denature them into linear chains and impart a uniform negative charge-to-mass ratio, ensuring separation is predominantly based on size.
Visualizing Separated Molecules
After electrophoretic separation, the typically invisible molecules must be made visible for analysis. This is achieved through staining techniques. For DNA and RNA, a common method involves staining the gel with a fluorescent dye such as ethidium bromide (EtBr) or safer alternatives like SYBR Safe. Ethidium bromide intercalates between nucleic acid base pairs. When exposed to ultraviolet (UV) light, the DNA-bound dye fluoresces, allowing distinct bands of separated DNA or RNA to be observed.
For proteins, common staining agents include Coomassie Brilliant Blue or silver stains. Coomassie blue dye binds to proteins. After the gel is soaked in the dye solution, excess dye is washed away, revealing the separated protein bands as blue lines against a clear background. This visualization step confirms the presence and relative sizes of the separated molecules.