Gel electrophoresis is a fundamental technique used across biology and chemistry to separate large molecules like DNA, RNA, and proteins. This separation is achieved by applying an electric field to a gel matrix, which acts as a molecular sieve. The visual output is an image displaying the separated molecules as distinct bands. Interpreting these resulting patterns allows researchers to determine the size, quantity, and sometimes the purity of the molecules within a given sample.
The Anatomy of a Gel
The foundation of interpreting any gel image begins with understanding its physical layout and how the molecules move through it. Samples are loaded into small indentations at one end of the gel slab, known as wells. Each well corresponds to a vertical track or lane that the sample travels down as the electric current is applied.
Molecules, which are typically negatively charged, migrate through the gel matrix from the negative electrode toward the positive electrode. This movement is differential, meaning that smaller molecules navigate the gel’s mesh-like structure more quickly and travel farther. Conversely, larger molecules encounter more resistance and remain closer to the starting well.
One lane is always dedicated to a molecular ladder, also known as a size marker. This marker is a pre-mixed solution containing molecules of known, standardized sizes. The ladder functions as an essential reference scale to determine the size of unknown molecules in the adjacent sample lanes.
Determining Fragment Size Using Molecular Ladders
The main goal of gel electrophoresis is to determine the size of the separated molecules by comparing them to the molecular ladder. To estimate the size of an unknown band, align its position horizontally with the bands in the adjacent ladder lane. If a sample band aligns exactly with a specific ladder band, the size of the unknown molecule is the same as the known size of that ladder fragment.
The relationship between the distance a molecule travels and its size is not a simple linear measurement. Migration distance is inversely proportional to the logarithm of the molecule’s size. This non-linear separation means that smaller bands at the bottom of the gel are spaced closer together than larger bands near the top. This principle makes the visual comparison to the ladder bands the most practical method for size estimation.
Before concluding size determination, control samples must be examined to confirm the procedure worked correctly. Positive controls, which contain a known molecule, should display a band at the expected size, validating the reagents and separation conditions. Negative controls, which contain no target molecule, should show no bands, confirming that the observed results are not due to contamination or artifacts. Validating these controls ensures that size measurements on experimental samples are reliable and accurate.
Interpreting Band Presence and Intensity
Beyond simply measuring size, the presence or absence of a band provides a qualitative conclusion about the sample. A clear, distinct band in a sample lane indicates that the target molecule, such as a specific DNA fragment, was successfully isolated or amplified during the experiment. Conversely, the absence of an expected band suggests the target molecule was not present in the original sample or that a step in the laboratory procedure failed.
The visual brightness and thickness of a band are used for semi-quantitative assessment, providing an estimate of the molecule’s concentration. A band that is thick and appears bright suggests a high concentration of that molecule is present in the sample. A faint, thin band indicates a lower abundance of the corresponding molecule.
This visual assessment is considered semi-quantitative because the amount of staining dye bound to the molecule is roughly proportional to the total amount of the molecule present. For precise quantification, specialized software is used to measure the light emitted or absorbed by the band, which correlates directly to the sample concentration. Observing multiple bands within a single lane can suggest several possibilities, including the sample containing a mixture of different sized target molecules, the presence of contamination, or the existence of different structural forms of the same molecule.
Recognizing and Troubleshooting Abnormal Patterns
The resulting gel image sometimes displays patterns that deviate from the expected clear, sharp bands, suggesting an issue with the sample or the separation process.
Smearing
One common abnormality is smearing, which appears as a continuous blur or streak of signal rather than distinct, focused horizontal bands. Smearing often results from the sample being overloaded into the well, which exceeds the gel’s separation capacity. It can also be caused by poor sample quality, such as molecule degradation or contamination by excessive salts or proteins. When a sample degrades, molecules break down into many fragments of varying sizes, which then migrate across the gel to create the blurred pattern. To correct this, scientists reduce the amount of sample loaded or ensure the sample is purified before running the gel.
Warping
Another recognizable distortion is the “smiling” or “frowning” effect, where the bands curve upward or downward at the edges of the gel. This warping is caused by uneven thermal distribution or voltage across the gel during the run, where the center heats up more than the edges. Running the gel at a lower voltage or in a cold environment minimizes this heat difference and restores a flat migration pattern.