Smeary bands on your SDS-PAGE gel are cringeworthy.
Not least because running an SDS-PAGE takes a little while and always seems to be the last thing you do. Bum note to end the day on when your gel doesn’t answer your question!
And getting a smeary band usually means you can’t answer the question you want to. Great.
This article explains a neat trick—using Bis-Tris gels to get high-resolution and sharper bands.
We’ll discuss the advantages of using Bis-Tris gels, consider when it makes sense, and explain how to cast them.
What Is a Bis-Tris Gel?
Bis-Tris gels are another type of gel you can use in Polyacrylamide Gel Electrophoresis (PAGE), along with regular SDS-PAGE gels and native PAGE gels, to separate proteins.
The pKa of Bis-Tris (technically called Bis-Tris methane) is 6.5 at 25°C, compared to a pKa of 8.1 for regular tris base.
The optimal buffering range for Bis-Tris gels is ~ 5.8–7.2
Thus, Bis-Tris gels are run at neutral or slightly acidic pHs, in contrast to the alkaline conditions used in conventional SDS-PAGE gels, making them suitable for separating proteins that are more stable or better resolved at this pH.
Acidic conditions can also help suppress cysteine reoxidation, preventing proteins from cross-linking via disulfide on the gel, but of course, you can always include a reducing agent to prevent this (see later).
Just beware that Bis-Tris is a chelating agent and binds strongly to common metal cations you use during protein expression and analysis such as zinc, calcium, cobalt, and nickel.
Advantages of Bis-Tris Gels
Although using Bis-Tris instead of tris makes your gels more expensive to prepare, they have two main:
- You get better resolution, sharpening up your protein bands
- They generally exhibit lower background staining than regular gels
It doesn’t sound like much but taken together, they can have a major beneficial impact on your experiments and analysis, especially if you consider how much analysis we use PAGE for.
When Is it a Good Idea to Use Them?
If you think about the advantages listed above, it might be a good idea to use Bis-Tris gels when:
- You intend to do a Western blot
- You are studying different protein forms that all have similar sizes
- You need to resolve a bunch of low molecular-weight bands
- Your sample is notorious for producing smeary bands (e.g., hydrophobic proteins)
- You need a really pretty image of a gel with nice sharp bands
If you want to see the process of doing a Western blot using a Bis-Tris gel result, or generally see how other labs are setting up their Bis-Tris gel system, check out this paper in the Journal of Visual Experiments. [1]
Your Bis-Tris Gel Recipe
Here’s how to make a Bis-Tris gel. The method is not very different from the conventional methods of casting protein gels.
Simply replace the tris buffer in the resolving gel with the Bis-Tris buffer at pH 6.4 (or whatever pH you need so long as it’s within the aforementioned buffering range).
You can use a lower concentration of Bis-Tris though compared to the concentration of tris you would normally use. Around 30% less to be precise.
Why?
It has to do with the buffering range of Bis-Tris mentioned above. Its effective buffering range is much narrower than that of tris (pH 7–9), so you need fewer Bis-Tris molecules to maintain the pH.
You’ll want to keep regular tris in your stacking gel (usually present at around pH 6.8) as we still require the same chemical environment to stack the proteins in the sample matrix before resolving them.
Table 1 below gives you a handy Bis-Tris gel recipe.
Table 1. Bis-Tris gel recipe.
Component | Amount for X % resolving gel | Amount for stacking gel |
Acrylamide, 30% | (0.5 x X) mL | 1.98 mL |
Tris, 0.5 M, pH 6.8 | 0 mL | 3.78 mL |
Tris, 1.0 M, pH 6.4 | 3.75 mL | 0 mL |
SDS, 10% w/v | 150 µL | 150 µL |
H2O
| 11.02 – (0.5 x X) mL | 9 mL |
TEMED | 7.5 µL | 15 µL |
APS, 10% w/v | 75 µL | 75 µL |
Total volume mL | 15 mL | 15 mL |
Note that w/v means weight by volume. |
And Table 2 tells you what percentage gel to use (how much acrylamide to include) depending on the molecular weight (MW) of your target protein.
Table 2. Summary of what protein sizes SDS-PAGE gels can resolve when prepared at 8–20% acrylamide.
Size of protein (kDa) | % acrylamide in resolving gel |
4–40 | 20 |
12–45 | 15 |
10–70 | 12.5 |
15–100 | 10 |
25-200 | 8 |
Bitesize Bio has a handy gel casting protocol, which you can read here.
Include MES or MOPs In Your Running Buffer
Another key difference when running Bis-Tris gels is that you include MES or MOPS in your running buffer instead of glycine.
MES or MOPS anions (the negatively charged ones) serve as the trailing ions in the running buffer system, while the tris cation (positively charged) serves as the leading ion.
If you’re unsure what “leading” and “trailing” ions mean in SDS-PAGE parlance, you can read about how SDS-PAGE works here. Anyway:
- Use MES when you want to separate low molecular weight proteins (≤50kDa)
- Use MOPs when you want to separate high molecular weight proteins (≥50kDa)
The following table tells you how to prepare these buffers.
Table 3. Recipe for MES and MOPs running buffers for SDS-PAGE using Bis-Tris gels.
Ingredient | 5x Low MW Running Buffer | 5x High MW Running Buffer |
Tris | 250 mM | 250 mM |
EDTA | 5 mM | 5 mM |
SDS | 0.5 % w/v | 0.5 % w/v |
MES | 250 mM | — |
MOPS | — | 250 mM |
Note that if you are preparing these buffer stocks for an entire lab, you can make them at 20x strength by multiplying everything by 4—they will last a bit longer.
Add sodium bisulfite, a mild reducing agent, to a concentration of 5 mM in the running buffer before each run. It’s a good idea to do this from a 1M stock.
Set up your SDS-PAGE system with your Bis-Tris gels exactly as you would for any other SDS-PAGE and run them at a constant voltage of 150V.
Other than that—everything is the same. Read this article if you need detailed advice on how to run your electrophoresis.
Taking the Bis. Making Your Own Bis-tris Gels Summarized
And that’s Bis-Tris gels in your experimental repertoire. You can break them out when you know you need an important gel or if you start working with a sample that’s tricky to resolve.
If you want to dive deeper into buffers and how they work, check out our webinar “Everything You Need to Know About Buffers.”
Originally published on September 12, 2008. Updated and revised on July 2016 and October 2024.
Reference
1. Penna A, Cahalan M. (2007). Western Blotting using the Invitrogen NuPage Novex Bis Tris minigels. J Vis Exp. 7:264