Gel electrophoresis is a fundamental laboratory technique used to separate large biological molecules, such as DNA fragments or proteins, based on their physical properties. The process involves applying an electrical current to move these molecules through a porous, jelly-like matrix, effectively acting as a molecular sieve. Interpreting the resulting pattern of separated molecules, which appear as distinct lines or “bands,” is crucial for drawing conclusions about a sample’s composition. This interpretation relies on understanding how these bands form, how a reference scale is used, and what certain irregularities in the pattern may signify.
The Mechanism of Band Formation
The separation of molecules in gel electrophoresis is driven by an electrical field applied across the gel, which is submerged in a conductive buffer solution. Biological molecules like DNA and RNA possess an inherent negative electrical charge due to their phosphate backbones. Therefore, they are loaded into wells at the negative end and pulled toward the positive electrode. The gel itself, typically made of agarose or polyacrylamide, forms a microscopic meshwork of pores that acts as a physical barrier.
As the charged molecules move through this mesh, their rate of travel is determined primarily by their size. Smaller molecules navigate the gel’s pores quickly, allowing them to travel farther down the gel toward the positive end. Conversely, larger molecules encounter more resistance and become tangled in the mesh, causing them to move slower and remain closer to the starting well. This results in a separation of the sample into discrete bands, with each band representing a collection of molecules of roughly the same size.
Using the Standard Ladder for Size Determination
To translate a band’s position on the gel into a meaningful size measurement, a molecular weight marker, commonly called a “ladder,” is run in a separate lane alongside the unknown samples. This ladder is a pre-mixed solution containing multiple fragments of the same type of molecule—DNA, RNA, or protein—each with a precisely known size. For DNA, fragment sizes are measured in base pairs (bp) or kilobases (kb), while protein sizes are measured in kilodaltons (kDa).
Size determination involves a direct visual comparison of the sample bands to the corresponding bands in the ladder. For example, if an unknown DNA band aligns horizontally with a band in the ladder labeled “500 bp,” the unknown fragment is estimated to be 500 base pairs long. Because the relationship between molecular size and distance migrated is not perfectly linear, particularly for larger molecules, this comparison provides a reliable estimate rather than an absolute value. To improve accuracy, researchers ensure the expected size of their target molecule falls between two known ladder bands.
Interpreting Band Intensity and Concentration
Beyond molecular size, the quantity of the separated molecule is inferred from the band’s intensity. The brightness or thickness of a band correlates directly with the amount of the molecule present in that specific fragment size. A thick, bright band indicates a high concentration of that particular molecule size in the original sample.
Conversely, a faint or thin band suggests that the sample contained only a small amount of that molecule. This visual assessment of intensity is most useful for making comparative judgments between different lanes or bands on the same gel. For instance, a brighter band in one lane compared to the same-sized band in another indicates a higher relative abundance of the target molecule in the first sample. This qualitative evaluation of concentration is a standard way to assess the success of an experiment, such as the yield from a DNA amplification reaction.
Understanding Abnormal or Unexpected Results
While a successful separation yields sharp, distinct bands, abnormal patterns are common and provide important troubleshooting information. One frequent issue is “smearing,” where the signal appears as a blurred vertical streak rather than a clean line. Smearing indicates that the molecules have been degraded into non-uniform fragments, or that the sample was overloaded, causing the concentration to exceed the gel’s capacity to separate the fragments.
The presence of multiple, closely spaced bands when only a single band is expected can signal a problem or reveal complexity in the sample. This may be due to contamination, such as the co-purification of unwanted nucleic acids or proteins. In the case of DNA, multiple bands can represent different structural forms of the same molecule, such as supercoiled, nicked, or linear plasmid DNA, which migrate at different speeds. The absence of a band in a lane suggests the target molecule was not present in the original sample or that a procedural error, such as a failure in sample preparation or staining, occurred.