Gel electrophoresis is a standard laboratory procedure used to separate large biological molecules, such as DNA, RNA, and proteins, based on their size and electrical charge. An electrical field pulls charged molecules through a porous gel matrix; smaller molecules travel faster and farther than larger ones. The desired outcome is a pattern of sharp, distinct lines, or bands, where molecules of uniform size have collected. Smearing occurs when these bands appear as diffuse, elongated trails or hazy patterns instead of clear lines. This loss of resolution makes the results difficult to interpret because the different size classes of molecules are not cleanly separated.
Sample Integrity and Preparation Errors
Smearing often originates from problems with the sample before it enters the gel matrix. A primary cause is the breakdown or partial degradation of the target molecules. For DNA or RNA, active nucleases (enzymes that cleave nucleic acids) break uniform molecules into a continuum of smaller fragments, which migrate as an indistinguishable trail rather than a single band. Similarly, active proteases digest proteins into many different peptide sizes, resulting in a diffuse pattern.
Mechanical stress is another factor that damages samples, especially large DNA molecules. Vigorous mixing, excessive vortexing, or forcing a viscous solution through a small-gauge needle can physically shear the DNA strands. This action breaks the long, uniform molecules into random, smaller fragments, mimicking enzymatic degradation and leading to a smear across the lane.
Chemical contaminants within the sample buffer can also interfere with proper migration. Residual salts or high concentrations of chelating agents alter the sample’s overall conductivity and ionic strength, locally disrupting the electrical field when loaded. Incorrect pH levels or certain detergents can affect the molecule’s net charge or conformation, causing molecules to adopt varying shapes. If molecules do not maintain a uniform structure or charge, they separate poorly and appear as a smear.
Running Buffer and Electrical Current Issues
The environmental conditions during the run influence the sharpness of the final bands. A significant cause of smearing is excessive voltage, which leads to Joule heating. Electrical resistance converts energy into heat, and high voltage dramatically increases this thermal output throughout the gel and buffer.
Excessive heat causes molecules to undergo thermal diffusion, moving randomly outside the intended migration path. Instead of maintaining a tight band front, molecules spread out perpendicular to the direction of migration, resulting in a diffuse smear. For DNA analysis, localized heating can also cause the double helix to partially melt or denature, changing its structure and slowing migration.
The quality and concentration of the running buffer directly impact the consistency of the electrical field. If the buffer is old or reused, its ionic components become exhausted or depleted. An exhausted buffer cannot maintain a stable pH or consistent conductivity, leading to changes in resistance and inconsistent migration speeds across the gel.
Improper preparation, such as over-diluting the buffer or neglecting to cover the gel fully, creates a non-uniform electrical field. When resistance changes dramatically across the gel, molecules migrate at different rates, disrupting the separation process. Maintaining the correct concentration and sufficient buffer volume guarantees a stable electrical current and consistent migration.
Gel Matrix Quality and Loading Density
The physical properties of the gel matrix and the amount of sample loaded affect separation effectiveness. Loading too much sample into a single well, known as overloading, often results in significant smearing. When molecule concentration exceeds the capacity of the gel’s pores to resolve them, the molecules aggregate or interact with each other.
This aggregation causes the sample to travel through the gel as an intertwined mass rather than as discrete entities. The resulting pattern is typically a vertical streak or a heavy, unresolved smear concentrated near the top of the lane. Proper quantification of the sample before loading ensures the amount is within the gel’s separation limits.
Physical imperfections in the gel structure can also compromise band integrity. Inconsistent gel polymerization results in localized areas with different pore sizes. These variations cause molecules to migrate at different speeds within the same lane, resulting in band distortion or smearing.
The wells, formed by the comb, must be clean and intact for the sample to enter the matrix uniformly. If wells are damaged, contain air bubbles, or have residual debris, the sample will not enter the gel in a tight line. This initial spreading of the sample is magnified throughout the run, leading to a smeared appearance.