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Tris-Glycine vs. Bis-Tris Gel Chemistry
March 5th, 2019
Tris-Glycine Chemistry
The Tris-Glycine gel formulation, based on the Laemmli system, is the simplest and most widely used system for separating a broad range of proteins by SDS PAGE and native PAGE (i.e., without SDS or alternative denaturant). The system utilizes two Tris-Glycine gels; the first is a stacking gel that helps focus the proteins into sharp bands at the beginning of the electrophoretic run; and the second is a resolving gel that separates the proteins based on their size.
The buffer system used is known as a discontinuous buffer system, which means that the pH and ionic strength of the buffer used for running the gel is more basic (i.e., more alkaline) compared with the buffers used in both the stacking and resolving gels. During a run the gel chloride ions move (migrate) towards the anode and away from the protein being electrophoresed, leaving the glycine ions trailing behind. Conversely, the Tris ions in the gel migrate towards the cathode, passing the protein on its way to the anode. Consequently, the protein becomes exposed to both Tris and glycine ions. However, for native PAGE, the pI of glycine (~6) is close to that of the stacking gel buffer, which means that glycine can help the protein to migrate only if the pI of the protein is lower than the pH of the stacking gel buffer, and the migration velocity will also depend on protein size, net charge and mass to charge ratio.
Tris-Glycine chemistry gels are also known for their poor band resolution. The operating pH, which is the actual pH during the run, is dissimilar when compared with the buffer pH at the start of the run and is highly alkaline. This can compromise protein separation and lead to band distortion, loss of resolution, artefact bands and problems with downstream applications (e.g. Mass spectrometry) and / or analysis.
During the time period of an electrophoretic run there is also a risk for glycine ions and the amine / thiol (sulfhydryl) groups of proteins to react with free nonpolymerized acrylamide, known as the Michael addition. This is a particular risk with handcast gels where there is a greater risk of gel nonhomogeneity. The concentration of free nonpolymerized acrylamide can often be much higher in handcast gels compared with precast gels.
Bis-Tris Gel Chemistry
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, it is the chloride ions that migrate to the anode, instead of glycine ions. The protein therefore becomes exposed to Bis-Tris, MES or MOPS ions and there is diminished reactivity of the amino and thiol (sulfhydryl) groups under such near neutral pH conditions. The pH of the sample buffer is 8.5 to avoid acidification during heating. Protein stability during electrophoresis at neutral pH is improved, resulting in sharper band resolution and accuracy.
The Bis-Tris gel formulation and pH of the gel, running and sample buffers has 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., 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 should not be compared with those obtained from Tris-Glycine gels, as the running buffers are different.
MES vs. MOPS Running Buffer for Bis-Tris Gels
Bis-Tris gels are compatible with MES or MOPs running buffer, but each can provide 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 Differences Between Tris-Glycine and Bis-Tris Gels
| Tris-Glycine Gels | Bis-Tris Gels | |
| Buffer system | Discontinuous | Discontinuous |
| pH Gel Buffer | ~8.3 | 6.4 |
| pH Sample buffer | 5.2 | 8.5 |
| pH Running Buffer | ~8.3 | 7.3–7.7 |
| Ions | • Tris+ • Gly- • SDS | • Tris+ • MES- • MOPS- • SDS |
| 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 |
Expedeon RunBlue™ Bis-Tris Precast Gels
Our RunBlue™ Bis-Tris Precast Gels are based on the Bis-Tris neutral gel buffer system. They have been developed to achieve exceptional reproducibility and resolution with highly comparable results to other commercial Bis-Tris gels. Expedeon 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. 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. There is no workflow disruption, no downtime, and no transition period.
Migration Chart for Expedeon’s RunBlue™ Bis-Tris Precast Gel with MES / MOPS Buffer
References:
- Graham, DRM., Garnham, CP., Fu, Q., et al. Improvements in two-dimensional gel electrophoresis by utilizing a low cost “in-house” neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteomics.2005:5(9);2309–2314. doi:10.1002/pmic.200401249
- Hachmann, JP., Anshey, JW. Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH. Anal Biochem.2005:342;237–245.
- Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature.1970:227;680–685.
- Moos Jr., M., Nguyen, NY., Liu, T-Y. Reproducible high yield sequencing of protein electrophoretically separated and transferred to an inert support. J. Biol. Chem.1988:263;6005–6008.
- Niepmann, M. Discontinuous native protein gel electrophoresis: pros and cons. Expert Rev Proteomics.2007;4:355–361.
