Gel electrophoresis separates large molecules like DNA, RNA, and proteins based on size and electrical charge using an electric current passed through a porous gel matrix. The surrounding buffer solution is essential, acting as the operational environment. It provides the necessary medium to conduct electricity while protecting the sample molecules from degradation. Without this liquid, the electrical circuit would be incomplete, compromising accurate separation.
The Dual Role of Conduction and pH Stabilization
The buffer’s primary function is to maintain electrical conductivity by supplying the electrolyte ions needed to carry the current between the electrodes. This flow of ions generates the electrical force that drives charged macromolecules through the gel matrix. If the buffer’s ionic strength (the concentration of conducting ions) is too low, the electrical resistance becomes too high, resulting in an insufficient or unstable current.
The second function is to stabilize the \(\text{pH}\) of the system, known as buffering capacity. Electrical current causes electrolysis, breaking down water molecules to form \(\text{H}^+\) and \(\text{OH}^-\) ions at the electrodes. This rapidly increases acidity and basicity, which would quickly alter the electrical charge of the sample molecules. The buffer, composed of a weak acid and its conjugate base, resists these \(\text{pH}\) shifts by absorbing or releasing \(\text{H}^+\) ions. Maintaining a stable \(\text{pH}\) ensures consistent migration speed and separation accuracy.
Preventing Sample Degradation and Maintaining Charge
The stable \(\text{pH}\) environment maintains the consistent electrical charge of the molecules being separated. For nucleic acids like DNA and RNA, a stable, slightly alkaline \(\text{pH}\) ensures they remain negatively charged and move predictably toward the positive electrode. If the \(\text{pH}\) drops, the molecule’s net negative charge is reduced, causing it to slow down or stop migrating.
For proteins, which are amphoteric, \(\text{pH}\) stability is more complex because their net charge depends highly on the surrounding environment. If the \(\text{pH}\) shifts to the protein’s isoelectric point (\(\text{pI}\)), its net charge becomes zero, causing it to cease movement or precipitate. Additionally, the buffer often includes chemicals like EDTA (ethylenediaminetetraacetic acid) to prevent sample degradation. EDTA works by chelating divalent metal ions, which are necessary cofactors for nucleases that would otherwise digest DNA or RNA samples.
Selecting the Right Buffer Formulation
The choice of buffer depends on the specific molecule being separated and the desired experimental outcome. The two most common buffer systems for nucleic acid electrophoresis are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). Both use Tris for \(\text{pH}\) stability and EDTA for sample protection, but their differing secondary ionic components affect performance.
Tris-Acetate-EDTA (TAE)
TAE buffer incorporates acetate ions, resulting in lower buffering capacity and lower ionic strength compared to TBE. This makes it more susceptible to \(\text{pH}\) changes during long runs. However, its lower ionic strength allows larger DNA fragments (greater than 12-15 kilobases) to migrate more efficiently. TAE is often preferred for downstream applications like cloning.
Tris-Borate-EDTA (TBE)
TBE buffer contains borate ions, offering a higher buffering capacity. It is better suited for separating smaller DNA fragments (less than 1,000 base pairs). TBE provides sharper band resolution due to its higher ionic strength.
What Happens When the Buffer Fails
A failure in the buffer system leads to easily identifiable problems with separation results. If the buffer concentration is too high, the system has low electrical resistance, causing excessive current flow. This high current generates massive heat (Joule heating), which can melt the gel matrix or damage samples. This often results in distorted, “smiling” bands where the center of the gel runs faster than the edges.
Conversely, if the buffer concentration is too low, high resistance causes the current to be unstable or slow, extending the run time and resulting in poor band resolution. The buffer can also become depleted after multiple uses, reducing its buffering capacity and allowing \(\text{pH}\) shifts. These shifts cause bands to smear or become fuzzy instead of remaining sharp. The most common failure occurs when the buffer level drops below the gel surface, breaking the electrical circuit and stopping migration.