The Resolving Solution can be diluted with deionized water to the desired acrylamide percentage, from 8% - 15%. The kit includes a 20% acrylamide Resolving Solution to form the resolving portion of an acrylamide gel. The TurboMix В® Bis-Tris Gel Casting Kit eliminates the variability and time-consuming nature of hand casting gels by supplying pre-mixed buffer and acrylamide solutions. Variation in gel buffer preparation can also lead to inconsistent gel performance and quality. Hand cast polyacrylamide gels are inexpensive compared to precast gels however, preparation of reagents and casting equipment can be tedious and time consuming. 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. This preparation maintains protein integrity by minimizing Asp-Pro peptide bond cleavage. Conversely, Bis-Tris gels use an LDS sample buffer that maintains an alkaline pH during sample preparation and does not require heating above 70 вЊ to fully denature proteins.

This leads to apparent protein degradation products during electrophoresis. Heating Laemmli buffer to 100 вЊ 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).

Samples are then boiled at 100 вЊ to help facilitate denaturation. In Tris-Glycine gels, Laemmli buffer is typically used to denature and coat proteins in negatively charged SDS ions. It is also important to consider the type of sample buffer used during protein preparation. 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. 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. Bis-Tris gels also have a longer shelf life than Tris-Glycine gels, which begin to hydrolyze over time. This leads to sharper protein band resolution and accuracy. Bis-Tris gels operate at a neutral pH which minimizes protein modification and promotes protein stability during the gel run. In contrast, Bis-Tris gels use Bis-Tris and HCl in the gel buffer and MOPS or MES in the running buffer. As a result, protein bands may be distorted or lose resolution in Tris-Glycine gels. Tris-Glycine gels operate in a highly alkaline environment which can lead to undesirable protein modifications like deamination and alkylation. 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. The difference in electrophoretic mobility between these two ions forms a moving voltage gradient which proteins travel through. PAGE uses a discontinuous buffer system, wherein the gel buffer ion differs from the running buffer ion. Small pores are desirable for separating low molecular weight proteins. Acrylamide concentration is linearly related to pore size a greater concentration of acrylamide will lead to smaller pores within the gel. The resolving gel is a higher percentage acrylamide that separates proteins by size. 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. Polyacrylamide gels are composed of two parts: a stacking portion and a resolving portion. Together, acrylamide and bis-acrylamide form pores through which proteins can migrate. Polyacrylamide gels are formed through the polymerization of acrylamide monomers and bis-acrylamide crosslinking molecules. Polyacrylamide gel electrophoresis (PAGE) is a foundational technique used to separate proteins and other macromolecules by their electrophoretic mobility. Polyacrylamide gel electrophoresis (PAGE)

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