Polyacrylamide gel electrophoresis (PAGE) is a foundational technique used to separate proteins and other macromolecules by their electrophoretic mobility. Polyacrylamide gels are formed through the polymerization of acrylamide monomers and bis-acrylamide crosslinking molecules. Together, acrylamide and bis-acrylamide form pores through which proteins can migrate.
Polyacrylamide gels are composed of two parts: a stacking portion and a resolving portion. The stacking portion of the gel is a low percentage acrylamide that serves to concentrate samples into a single band before entering the resolving gel. The resolving gel is a higher percentage acrylamide that separates proteins by size. Acrylamide concentration is linearly related to pore size; a greater concentration of acrylamide will lead to smaller pores within the gel. Small pores are desirable for separating low molecular weight proteins.
PAGE uses a discontinuous buffer system, wherein the gel buffer ion differs from the running buffer ion. The difference in electrophoretic mobility between these two ions forms a moving voltage gradient which proteins travel through. Tris-Glycine gel chemistry is the most commonly used PAGE system, which uses gels composed of Tris-HCl and running buffer composed of Tris base and glycine. Tris-Glycine gels operate in a highly alkaline environment which can lead to undesirable protein modifications like deamination and alkylation. As a result, protein bands may be distorted or lose resolution in Tris-Glycine gels.
In contrast, Bis-Tris gels use Bis-Tris and HCl in the gel buffer and MOPS or MES in the running buffer. Bis-Tris gels operate at a neutral pH which minimizes protein modification and promotes protein stability during the gel run. This leads to sharper protein band resolution and accuracy. Bis-Tris gels also have a longer shelf life than Tris-Glycine gels, which begin to hydrolyze over time. Bis-Tris gels have the flexibility to be combined with either MOPS- or MES-based running buffer; the difference in migration between these two ions results in different protein separation ranges. MES should be used when the protein of interest is small (<50 kDa) while MOPS should be used to resolve mid- to large-sized proteins.
It is also important to consider the type of sample buffer used during protein preparation. In Tris-Glycine gels, Laemmli buffer is typically used to denature and coat proteins in negatively charged SDS ions. Samples are then boiled at 100 °C to help facilitate denaturation. Heating Laemmli buffer to 100 °C causes the pH to become highly acidic and this combination of heat and acidity has been shown to cause protein cleavage preferentially at Asp-Pro peptide bonds (Rittenhouse and Marcus, 1984). This leads to apparent protein degradation products during electrophoresis. Conversely, Bis-Tris gels use an LDS sample buffer that maintains an alkaline pH during sample preparation and does not require heating above 70 °C to fully denature proteins. This preparation maintains protein integrity by minimizing Asp-Pro peptide bond cleavage.
Figure 1.Comparison of Tris-Glycine (left) and Bis-Tris (right) gels. Tris-Glycine and Bis-Tris gels were hand-cast with 12% acrylamide and allowed to polymerize overnight. The gels were loaded with identical E. coli lysate titrations (lanes 3-6), mPAGE® unstained protein standard (lanes 2 and 7), and mPAGE® color protein standard (lane 1). The gels were run in either Tris-Glycine or MOPS running buffer, stained with ReadyBlue™ protein gel stain for one hour, and destained with deionized water for one hour.
While precast polyacrylamide gels can be purchased for specialized purposes, such as analyzing proteins of different sizes on an acrylamide gradient, many researchers choose to cast their own polyacrylamide gels by hand. Hand cast polyacrylamide gels are inexpensive compared to precast gels; however, preparation of reagents and casting equipment can be tedious and time consuming. Variation in gel buffer preparation can also lead to inconsistent gel performance and quality.
The TurboMix™ Bis-Tris Gel Casting Kit eliminates the variability and time-consuming nature of hand casting gels by supplying pre-mixed buffer and acrylamide solutions. The kit includes a 20% acrylamide Resolving Solution to form the resolving portion of an acrylamide gel. The Resolving Solution can be diluted with deionized water to the desired acrylamide percentage, from 8% - 15%. The kit also includes a 4% acrylamide Stacking Solution to form the stacking portion of an acrylamide gel. These solutions are formulated to allow for a Quick Casting method with no polymerization wait time between pouring the resolving and stacking gels. Below is a demo video and brief protocol.
Table 2. Preparation of resolving solutions and stacking solutions for gel casting. Volumes listed are sufficient for casting one 7.4 x 8.2 cm mini-gel with 1 mm thickness and can be multiplied by n (desired number of gels) to cast multiple gels at once.