Tris-glycine gels are a foundational tool in molecular biology for separating proteins using a technique called polyacrylamide gel electrophoresis (PAGE). This method uses a gel matrix to sort proteins based on their size, providing a clear snapshot of the protein composition within a sample. This allows researchers to analyze everything from simple purified proteins to complex mixtures derived from cells and tissues.
The Chemical Composition
The gel itself is a matrix formed by the polymerization of acrylamide and a cross-linking agent, bis-acrylamide. By adjusting the concentration of acrylamide, scientists can change the pore size of this matrix. A higher percentage of acrylamide results in smaller pores for separating small proteins, while a lower percentage creates larger pores for resolving bigger proteins. This gel matrix is submerged in a running buffer that contains Tris and glycine.
Tris, or tris(hydroxymethyl)aminomethane, is the primary buffering agent. Its purpose is to maintain a stable pH throughout the electrophoretic run, ensuring consistent conditions. Glycine, an amino acid, serves as the “trailing ion” in this system. Its charge is dependent on the pH of its environment, a feature important for the separation process.
Sodium Dodecyl Sulfate (SDS) is added to the sample buffer and often the gel itself. This detergent unfolds complex three-dimensional proteins into linear chains and coats them with a uniform negative charge. This treatment ensures a protein’s original shape and charge do not influence its movement, meaning separation occurs based almost exclusively on its molecular weight.
The Separation Mechanism
The separation of proteins is achieved through a discontinuous buffer system, famously developed by Ulrich Laemmli. This system uses two different gel layers with distinct pH levels and pore sizes: a “stacking gel” on top and a “resolving gel” below. This dual-gel setup is fundamental to achieving the sharp, well-defined protein bands that characterize this technique.
The stacking gel has a lower acrylamide concentration, creating large pores, and a lower pH, typically around 6.8. Its function is not to separate the proteins but to concentrate them into a thin, focused band. When the electric current is applied, the SDS-coated, negatively charged proteins in the sample move into this gel. Here, they are sandwiched between highly mobile leading chloride ions and slower-moving trailing glycinate ions from the running buffer.
This “stack” of molecules moves through the stacking gel and reaches the interface of the resolving gel. The resolving gel has a higher pH, around 8.8, and a higher acrylamide concentration, resulting in smaller pores. The abrupt increase in pH causes the glycine molecules to become more negatively charged and accelerate, moving past the proteins. As the proteins enter the smaller-pored resolving gel, they are no longer concentrated and begin to separate based on size. Smaller proteins navigate the matrix more easily and travel farther, while larger proteins are impeded and move more slowly.
Practical Laboratory Applications
The primary application of Tris-glycine gels is Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The main purpose of SDS-PAGE is to determine the molecular weight of proteins. By running a sample alongside a “ladder” containing proteins of known molecular weights, researchers can estimate the size of their protein of interest by comparing its migration distance to the standards. This is a routine procedure for characterizing newly isolated proteins.
Another common use of this method is to assess the purity of a protein sample. For instance, after a series of purification steps, a scientist can run a small amount of the sample on a gel. A successful purification would show a single, strong band, indicating one dominant protein. The presence of multiple bands signifies that the sample still contains contaminants.
Running a Tris-glycine gel is often a preparatory step for other analytical techniques, with the most prominent being the Western blot. After proteins are separated by SDS-PAGE, they can be transferred from the gel onto a solid membrane. This membrane can then be probed with specific antibodies to detect the presence and quantity of a single target protein within the complex mixture.
Comparison with Other Gel Systems
While Tris-glycine is a widely used system, alternative gel chemistries exist for specific research needs. Bis-Tris gels, for example, operate at a more neutral pH (around 7.0) compared to the alkaline conditions of a running Tris-glycine gel. This neutral environment is gentler on proteins, minimizing the risk of chemical modifications like deamination. This makes Bis-Tris gels a better choice for sensitive downstream applications like mass spectrometry, and they also have a longer shelf life.
For the analysis of very small proteins and peptides, typically those under 20 kDa, Tris-Tricine gels are often preferred. In the standard Tris-glycine system, small proteins can migrate with the dye front, leading to poor resolution. The Tris-Tricine system modifies the buffer composition, replacing glycine with tricine as the trailing ion, which allows for better separation of these low-molecular-weight molecules.
Researchers looking to separate a very wide range of protein sizes in a single experiment can turn to gradient gels. Instead of a uniform acrylamide concentration, these gels have a gradient, for example, from 4% at the top to 20% at the bottom. This allows for the simultaneous resolution of both large and small proteins on one gel, offering a broader view than a single-percentage Tris-glycine gel.