Gel electrophoresis is a laboratory technique used to separate and analyze large biological molecules such as DNA, RNA, and proteins. This method provides a visual output, allowing researchers to examine these molecules, and is widely used in molecular biology and diagnostics. Interpreting electrophoresis results is essential for extracting meaningful information from experiments.
The Basics of Electrophoretic Separation
Electrophoresis works by applying an electric current to charged molecules within a porous gel matrix. Molecules migrate through the gel because they possess an electrical charge; for instance, DNA and RNA are negatively charged due to their phosphate backbones and move towards the positive electrode. The gel acts like a sieve, impeding the movement of larger molecules more than smaller ones. This causes molecules to separate primarily based on their size, with smaller molecules moving faster and traveling further.
The rate of migration is also influenced by the molecule’s charge and shape. Proteins are often treated with a denaturing agent like SDS to give them a uniform negative charge, allowing their separation primarily by size. This mechanism produces distinct bands on the gel, each representing a collection of molecules of a particular size or characteristic.
Elements of an Electrophoresis Gel
A developed electrophoresis gel presents several key visual components for interpretation. Samples are loaded into small indentations at one end of the gel called “wells.” From these wells, molecules migrate through distinct paths known as “lanes.” As the molecules separate, they form visible lines or spots called “bands.”
A molecular weight marker, or “ladder,” is typically loaded into one or more lanes. This ladder consists of molecules of known sizes, serving as a ruler to determine the size of unknown molecules. Comparing sample bands to the known bands of the ladder is a fundamental step in interpretation.
Interpreting Band Location and Intensity
The location of a band on an electrophoresis gel corresponds to the molecule’s size. Smaller molecules travel further down the gel, away from the wells, while larger molecules remain closer to the loading wells. To estimate the size of an unknown band, its migration distance is compared to the known sizes in the molecular ladder run on the same gel.
The intensity of a band indicates the quantity of the molecule present. A darker, thicker band suggests a higher concentration of that specific molecule. Conversely, a faint or thin band indicates a smaller amount. This visual assessment allows for qualitative comparisons of molecular quantities.
Recognizing Common Electrophoresis Patterns
Different types of molecules produce characteristic patterns on electrophoresis gels, providing insights into sample composition. For DNA analysis, PCR products appear as distinct bands at specific sizes. Genomic DNA often appears as a high molecular weight band or a dense smear near the loading well; if degraded, it might show a smear of smaller fragments. Plasmid DNA can exhibit multiple bands due to different conformations (supercoiled, relaxed circular, and linear forms), which migrate at different rates.
For RNA analysis, ribosomal RNA (rRNA) often appears as two distinct bands in eukaryotic samples: the 28S and 18S rRNA bands. Sharp rRNA bands, with the 28S band approximately twice as intense as the 18S band, indicate intact, high-quality RNA. Conversely, a smeared appearance or a ratio significantly less than 2:1 suggests RNA degradation. Messenger RNA (mRNA) typically appears as a smear across a range of sizes, representing a diverse population of transcripts.
Protein electrophoresis, using SDS-PAGE, separates proteins primarily by their molecular weight after denaturation. This technique yields specific protein bands, and their sizes are determined by comparing their migration to a protein ladder. The pattern of bands can reveal the presence of specific proteins, their purity, and their relative abundance.