Gel electrophoresis is a laboratory technique used to separate large biological molecules such as DNA, RNA, or proteins. This process relies on an electric current to move these molecules through a gel. The visible result appears as distinct lines within the gel, commonly referred to as “bands.” These bands provide information about the molecules within a sample.
How Bands Form
The formation of bands begins with a gel matrix, typically made from agarose or polyacrylamide, which acts like a sieve. Samples are loaded into wells at one end of this gel. An electric current is then applied across the gel, with one end having a negative charge and the other a positive charge.
Most biological molecules, like DNA and RNA, possess an inherent negative charge. When an electric field is introduced, these negatively charged molecules are repelled by the negative electrode and drawn towards the positive electrode. Smaller molecules move faster and farther through the porous gel than larger ones. This difference in speed causes molecules of similar size and charge to gather, forming visible bands at distinct positions within the gel.
What Molecules Are Represented by Bands
Bands represent groups of biological macromolecules, such as DNA, RNA, and proteins. For nucleic acids like DNA and RNA, separation is predominantly based on their size due to their uniform negative charge. Shorter DNA or RNA fragments will travel further through the gel, forming bands closer to the positive end, while longer fragments remain closer to the starting wells.
Proteins can also be separated using gel electrophoresis, though their separation often depends on both size and charge. To separate proteins primarily by size, they are typically treated with a chemical that gives them a uniform negative charge, allowing them to migrate similarly to DNA. Each band signifies a collection of molecules that are approximately the same size and, for proteins, share similar charge characteristics.
Interpreting the Information in Bands
The position of a band on a gel provides specific information about the size of the molecules it represents. Molecules that travel further down the gel are smaller, while those that remain closer to the starting well are larger. To accurately determine the size of the molecules in a sample, a “molecular weight marker” or “DNA ladder” is run in a separate lane alongside the samples. This ladder consists of molecules of known sizes, allowing for an estimation of the size of the unknown molecules by comparing their band positions. The intensity of a band indicates the relative quantity of molecules present in that specific size group. The presence or absence of specific bands is also significant, revealing whether particular molecules are present or missing from a sample.
Practical Applications of Bands
Gel electrophoresis bands are important across various scientific fields. In forensic science, the unique banding patterns of DNA can be used for DNA fingerprinting to identify individuals or establish paternity. Researchers use band analysis in gene expression studies to observe which genes are active and producing RNA in different conditions.
It is also widely applied in protein analysis, helping scientists determine the purity of samples or identify specific proteins. In medical diagnostics, observing certain banding patterns can help identify genetic mutations linked to diseases. It is routinely employed to verify the success of molecular biology experiments, such as polymerase chain reaction (PCR), by confirming the size and presence of amplified DNA fragments.