Straightening Out Messy Protein Gels  

Bis-Tris protein gels permit researchers to obtain high-resolution, publication-ready results.

The Scientist Creative Services Team in collaboration with MilliporeSigma
Sep 28, 2021

Protein analysis is an integral component of many research projects; understanding a protein of interest’s expression level or modification status can offer key mechanistic insights into the pathways that are active within a cell. To separate and analyze proteins, researchers most commonly use polyacrylamide gel electrophoresis (PAGE). While generations of scientists have used and improved protein gel electrophoresis, obtaining a clean protein gel with crisp bands remains a challenge.

To separate proteins by PAGE, scientists first choose between purchasing pre-cast gels or casting their own. While precast gels are convenient and show higher reproducibility rates than handmade gels, the latter gives researchers more flexibility to adjust a gel’s polyacrylamide concentrations to achieve optimal protein separation. Next, scientists must decide on a gel chemistry and buffer system for gel electrophoresis. Many researchers choose Tris-Glycine chemistry to separate proteins. In this method, scientists use Tris-HCl to create the protein gel while they incorporate Tris base and glycine into the running buffer. Under an electrical current, this chemical combination in the gel and accompanying buffer creates a voltage gradient that induces protein migration. 

A significant drawback of Tris-Glycine gels is variable pH; during electrophoresis the pH within the gel creates a highly alkaline environment. Elevated pH causes unwanted protein modifications, such as deamination or alkylation, that decrease sample integrity. In addition, alkaline conditions reduce a Tris-Glycine protein gel’s resolving power because they induce polyacrylamide hydrolyzation into polyacrylic acid. Both factors contribute to band distortion and poor resolution, known limitations of Tris-Glycine gels.

A second factor that decreases a Tris-Glycine gel’s resolution is the sample preparation method. To denature proteins and coat them with sodium dodecyl sulfate (SDS), scientists add Laemmli buffer to protein samples and boil them at 100 °C for up to ten minutes. At high temperature, Laemmli buffer becomes highly acidic. The combination of low pH and high heat causes protein cleavage at aspartate-proline peptide bonds, creating additional peptides that can confound data analysis. Therefore, researchers can take every measure to avoid protein degradation during sample preparation and still end up with smeared or additional bands on their western blots when using Laemmli buffer.  

Bis-Tris protein gel systems, which use Bis-Tris in the gel buffer and MES or MOPS in the running buffer, are a better option for protein analysis. Because the pH remains neutral during Bis-Tris gel electrophoresis, protein and gel stability is higher, resulting in protein gels and blots with crisper bands of higher resolution. In addition, Bis-Tris sample preparation requires scientists to heat samples to 70 °C in LDS buffer rather than Laemmli buffer, bypassing the boiling-induced peptide cleavage that is common for Tris-Glycine gels. As an additional advantage, scientists can increase their Bis-Tris gel’s resolving power by adjusting the type of running buffer; MES improves separation of smaller proteins whereas MOPS helps resolve high molecular weight proteins. Finally, Bis-Tris protein gels have a longer shelf-life and a shorter run-time compared to Tris-Glycine gels. 

MilliporeSigma created a wide variety of precast mPage® Bis-Tris protein gels that offer high performance, optimal electrophoretic separation, and improved resolution for the analysis of proteins ranging from 6 kDa to 400 kDa. Apart from increased resolution, these gels also allow scientists to load up to 80 µl sample, and so are ideally suited for low-abundance protein analysis and for protein purification.

For projects that require self-cast gels, MilliporeSigma developed the Turbomix Bis-Tris gel casting kit. To reduce variability, this kit comes with pre-mixed acrylamide solutions that scientists dilute to the desired percentage. The Turbomix gel casting method follows a simple one-step protocol that significantly speeds up the process. Indeed, this mix allows scientists to immediately pour the stacking gel on the resolving gel without waiting for the latter to polymerize. 

Researchers who are interested in comparing Tris-Glycine to Bis-Tris protein gels can request a variety of Bis-Tris-based protein gels or casting kits through MilliporeSigma’s sample program.