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Separating proteins on a gel offers key information regarding your protein of interest, but picking the right buffer system and gel chemistry can influence your results.
Learn how to pick the best combination for your target analysis with our guide to gel chemistry and buffer systems.
The Tris-Glycine gel formulation for gel electrophoresis is the simplest and most widely used system for separating a broad range of proteins using SDS PAGE or native PAGE (i.e., without SDS or alternative denaturant). Based on the Laemmli system it utilizes two Tris-Glycine gels; an initial stacking gel that focuses the proteins into a sharp band at the beginning of the electrophoretic run, followed by a resolving gel that separates the proteins based on their size.
This system uses a discontinuous buffer system, relies on the stacking effect of the boundary between the negative chloride ions at the leading edge and the trailing glycinate ions for proteins to migrate through the gel towards the anode at a rate dependent upon their molecular weight.
Tris-Glycine chemistry gels are known for their poor band resolution. Prepared at pH 8.6-8.8, the highly alkaline nature of these gels causes hydrolyzation of polyacrylamide to polyacrylic acid, which can compromise separation. The operating pH, which is the actual pH during the run, is can reach pH 9.5, further compromising protein separation and leading to band distortion, loss of resolution, artifact bands and problems with downstream applications (e.g. Mass spectrometry) and/or analysis.
During the time of the electrophoretic run there is also a risk for the glycine ions and the amine/thiol (sulfhydryl) groups of proteins to react with free non-polymerized acrylamide, known as the Michael addition. This is a particular risk with hand-cast gels where there is a greater risk of gel non-homogeneity. The concentration of free non-polymerized acrylamide can often be much higher in hand-cast gels compared with precast gels.
The conditions for electrophoresis (pH and buffers) are more favorable with Bis-Tris chemistry-based gels. These gels are HCI buffered and have a neutral operating pH. The running buffer can either be MES (50mM, with 50mM Tris) at pH 7.2 or MOPS (with Tris) at pH 7.7.
Compared to Tris-Glycine gels, these gels employ chloride as the leading ions and MES or MOPS as the trailing ions. With a sample buffer of pH 8.5, acidification of the polyacrylamide during running is reduced. Additionally, the neutral pH of the gel improves protein stability during running, resulting in sharper band resolution and accuracy.
The Bis-Tris gel formulation and pH of the gel, running and sample buffers have been shown to significantly reduce the risk of protein modifications such as deamination and alkylation. Furthermore, at nearly neutral pH, there is a significantly reduced risk for glycine ions and the amine/thiol (sulfhydryl) groups of proteins to react with free nonpolymerized acrylamide. Although there is the potential for this to occur, the half-life for reactive functional groups is much longer for Bis-Tris compared with Tris-Glycine (e.g., the half-life for protein sulfhydryl in Tris-Glycine with 10mM free acrylamide is 15 mins, compared with ~4 hours for a Bis-Tris gel with MES buffer).
A final consideration is that the gel patterns obtained from Bis-Tris gels are not directly comparable with those obtained from Tris-Glycine gels, due to the variation in the running buffers.
Bis-Tris gels are compatible with MES or MOPs running buffer, with each offering differential separation and resolution of proteins.
MES running buffer has been shown to better resolve small molecular weight proteins, whereas MOPS running buffer has been shown to better resolve medium-sized proteins.
MES has a lower pKa than MOPS, which enables gels with MES running buffer to run faster than gels with MOPS running buffer. The difference in ion migration affects stacking and results in a difference in protein separation range between these buffers.
The table below summarizes the differences between Tris-Glycine and Bis-Tris gels.
|Tris-Glycine gels||Bis-Tris gels|
|pH gel buffer||~8.3||6.4|
|pH sample buffer||5.2||8.5|
|pH running buffer||~8.3||7.3–7.7|
|Operating pH||9.5 (highly alkaline)||7.0 (neutral)|
|Protein stability during electrophoresis and band resolution /sharpness||Chemical alterations (e.g., deamination and alkylation) resulting in blurred or multiple bands and changes in electrophoretic capabilities||+++|
|Effect on proteins containing Asp–Pro bonds||Asp–Pro bonds can be reduced/cleaved when heated at 100°C in Laemmli sample buffer, pH 5.2||Disulfide (S–S) bonds are completely reduced under mild heating conditions (70°C for 10 minutes), whereas the Asp–Pro bonds remain intact|
|Effect on reducing agents (e.g., β-ME and DTT)||High pH --> risk of entry and migration with protein||Low pH prevents entry to the gel and migration with the protein|
|Gel redox state||Inconsistent --> reoxidation of reduced/cleaved S–S bonds for proteins with Cys residues||Consistent – reduced risk of reoxidation|
|Risk of Michael reaction leading to reactive functional groups||Yes (half-life: 15 mins)||Negligible (half-life: 4 hours)|
|Risk of pH-dependent methionine and tryptophan oxidation||Yes||No|
Our RunBlue™ Bis-Tris Precast Gels are based on the Bis-Tris neutral gel buffer system. Developed as an alternative to NuPAGE®Bis-Tris gels, they offer benefits such as comb/strip free design and tear-proof composition.
We have developed a unique physical induction technology that ensures homogenous polymerization across the whole gel, with no residual free acrylamide at the end of the polymerization process. Our gels deliver exceptional reproducibility and resolution with highly comparable results to other commercial Bis-Tris gels. RunBlue™ Precast Gel’s proprietary polymerization process results in more uniform gels between batches, with decreased variability and improved repeatability of results.
The gels are run with standard MES or MOPS buffers depending on the molecular weight range of proteins to be separated, providing better resolution while following the same migration profiles. You can also use the same LDS sample buffer, resulting in maximum efficacy with no workflow disruption, no downtime, and no batch-to-batch standardization.
Migration Chart for RunBlue™ Bis-Tris Precast Gel with MES / MOPS Buffer