A 10x Tris-buffered saline (TBS) stock solution is made by dissolving Tris base and sodium chloride in distilled or deionized water, adjusting the pH with hydrochloric acid, and bringing the total volume to one liter. The standard recipe calls for about 24 g of Tris base and 80 g of NaCl per liter, yielding concentrations of roughly 200 mM Tris and 1.37 M NaCl at the concentrated stock level. The process itself takes under an hour, but a few details, especially around pH adjustment and temperature, trip people up more often than you might expect.
Ingredients and Equipment
Before you start weighing anything, gather the following:
- Tris base: 24.2 g (molecular weight 121.14 g/mol). This gives 200 mM Tris in the final 10x stock. Some protocols round to 24 g, which makes virtually no difference at this concentration.
- Sodium chloride (NaCl): 80 g (molecular weight 58.44 g/mol). This produces approximately 1.37 M NaCl in the stock.
- Concentrated hydrochloric acid (HCl): Used to bring the pH down from the naturally alkaline Tris solution to the target range, typically pH 7.6. You can also use 6 M or 12 M HCl. Have more on hand than you think you need.
- Distilled or deionized water: Enough to reach a final volume of 1 liter.
- A calibrated pH meter: pH strips are not precise enough for buffer work.
- A magnetic stir plate and stir bar: For dissolving solids and mixing during pH adjustment.
- A graduated cylinder or volumetric flask: For accurate final volume.
Reagent-grade chemicals are the standard. Technical-grade Tris and NaCl can introduce trace contaminants that interfere with sensitive downstream assays like Western blots or immunohistochemistry.
Step-by-Step Preparation
Start by adding roughly 800 mL of distilled or deionized water to a clean beaker. You want headroom because you will be adjusting pH and topping off to 1 liter at the end, and adding HCl adds volume. Place the beaker on a magnetic stir plate and drop in a stir bar.
Weigh out 24.2 g of Tris base and 80 g of NaCl on an analytical balance. Add both to the beaker. Turn on the stir plate at a moderate speed. Tris base dissolves readily at room temperature, and NaCl dissolves quickly too, though the large quantity means you may need to wait a minute or two for it to fully go into solution. If undissolved crystals cling to the walls of the beaker, a gentle swirl or slight warming helps.
Once both solutes are completely dissolved, the solution will be quite alkaline, often reading above pH 10. This is normal. Tris base in water without acid is strongly basic. Slowly add concentrated HCl while stirring. The pH will drop steadily. As you approach pH 7.6, switch to smaller additions and wait for the meter to stabilize between each one. Overshooting the target pH is one of the most common mistakes in buffer preparation, and fixing it is annoying because adding Tris base back in changes your concentration.
Once the pH reads 7.6 at room temperature, transfer the solution to a graduated cylinder or volumetric flask and bring the total volume to exactly 1 liter with additional distilled or deionized water. Mix gently, then check the pH one more time. It may have shifted slightly with the dilution, and you can fine-tune it with a drop or two of HCl or a tiny amount of Tris base.
Why pH 7.6 and Not 7.4
You will see TBS recipes that target pH 7.4 and others that target pH 7.6. Both are used in real labs, and the choice often comes down to the specific protocol you are following. The 7.6 target is traditional for many immunoblotting and immunohistochemistry applications, while 7.4 aligns more closely with physiological pH and is common in cell-based work. If your protocol specifies a pH, use that number. If it does not, 7.6 is the safer default for most TBS applications.
One thing to keep in mind is that Tris buffers are sensitive to temperature in a way that catches people off guard. If you adjust the pH while the solution is warm and then store it at 4 °C, the pH at cold temperature will be measurably higher than what you set. The shift is roughly 0.03 pH units per degree Celsius for Tris-based buffers, which means a solution adjusted to pH 7.6 at 25 °C could read close to pH 8.0 at 4 °C. Research on buffer systems has confirmed that buffers containing amino groups, like Tris, are the most affected by temperature changes, with the pKa decreasing as temperature increases.1International Journal of Pharmaceutics. Buffer solutions in drug formulation and processing: How pKa values depend on temperature, pressure and ionic strength If your assay runs at a specific temperature, adjust the pH at that temperature or account for the drift.
Diluting to a 1x Working Solution
The whole point of a 10x stock is convenience: you make it once and dilute it many times. To prepare 1x TBS, mix one part 10x stock with nine parts distilled or deionized water. For a liter of working solution, that means 100 mL of stock topped off to 1,000 mL. For 500 mL, use 50 mL of stock and 450 mL of water.
Your final 1x solution will contain roughly 20 mM Tris and 137 mM NaCl, which closely mimics physiological salt concentrations. Check the pH of the diluted solution if your application is pH-sensitive. Dilution itself can shift the reading slightly, and the water you dilute into may have a different temperature than the stock, compounding the temperature-pH issue described above.
Making TBS-T by Adding Tween-20
For Western blotting and many immunoassays, you need TBS plus a small amount of the nonionic detergent Tween-20, commonly called TBS-T. The standard concentration of Tween-20 in the working solution is 0.1%, which translates to 1 mL of Tween-20 per liter of 1x TBS. Some protocols call for 0.05%, particularly when working with antibodies that have weaker affinity and might be washed off by stronger detergent concentrations.
Add the Tween-20 to the already-diluted 1x TBS, not to the 10x stock. Tween-20 is viscous and sticky, so pipetting it accurately can be frustrating. A common trick is to cut the tip of your pipette tip to widen the opening, or to use a positive-displacement pipette. Swirl or stir gently until the Tween-20 is fully incorporated. You will know it is mixed when the solution looks uniformly clear without oily streaks.
TBS-T should ideally be made fresh or within a day or two of use. Tween-20 can degrade over time, and microbial growth in a detergent-containing aqueous solution at room temperature is a real concern. If you notice cloudiness or particulates in your TBS-T, discard it and make a new batch.
Storage and Shelf Life
A properly prepared 10x TBS stock stored at room temperature in a clean, capped container will last for months. Some labs keep stocks for up to six months without issues. Autoclaving the solution after preparation extends its usability further by eliminating microbial contamination, though it is not strictly necessary for most applications.
The main threats to a stored TBS stock are microbial contamination and pH drift from dissolved carbon dioxide. COâ‚‚ from the atmosphere dissolves into aqueous solutions and forms carbonic acid, which gradually lowers the pH. A study examining Tris buffer stability found that COâ‚‚ infiltration and microbial respiration both contributed to measurable decreases in pH over storage time.2Ocean Science. Technical note: Stability of tris pH buffer in artificial seawater stored in bags Keeping the container tightly sealed reduces this problem. If your stock has been sitting open on a benchtop for days, check the pH before using it.
Refrigeration at 4 °C slows microbial growth but, as discussed earlier, shifts the apparent pH upward. If you store at 4 °C, let the stock equilibrate to room temperature and recheck the pH before use, or adjust the pH at 4 °C initially if the buffer will always be used cold.
TBS Versus PBS and When to Choose Each
Phosphate-buffered saline (PBS) is the other workhorse buffer in biology labs, and many newcomers wonder whether TBS and PBS are interchangeable. They often are, but there are specific situations where the choice matters.
TBS is the standard for immunohistochemistry and Western blotting when alkaline phosphatase-conjugated antibodies are involved. PBS contains phosphate ions, which inhibit alkaline phosphatase and can ruin your signal. If your detection system uses horseradish peroxidase (HRP) instead, PBS usually works fine, but many labs default to TBS for all immunoassays just to avoid having to think about which enzyme system they are using.
PBS has its own advantages. Its pH is less temperature-sensitive than Tris-based buffers, making it more stable across the temperature range you encounter in a typical experiment. It also better mimics the ionic environment of bodily fluids, which matters for cell culture and some in vivo applications. However, the phosphate ions in PBS can react with divalent cations like calcium and magnesium to form insoluble precipitates, which limits its usefulness in assays involving those ions.3Journal of Materials Science & Technology. New insights into the effect of Tris-HCl and Tris on corrosion of magnesium alloy in presence of bicarbonate, sulfate, hydrogen phosphate and dihydrogen phosphate ions TBS avoids this problem entirely because it contains no phosphate.
A simple rule of thumb: if the protocol says TBS, use TBS. If it says PBS, use PBS. If it does not specify, consider whether your detection system involves alkaline phosphatase (use TBS) or whether divalent cations are present in your sample (also use TBS). Otherwise, either buffer is usually acceptable.
Common Mistakes and How to Avoid Them
The most frequent error is confusing Tris base with Tris-HCl (also sold as Tris hydrochloride). These are different compounds with different molecular weights. Tris base has a molecular weight of 121.14, while Tris-HCl weighs 157.60. If you substitute one for the other without adjusting the mass, your Tris concentration will be wrong, and you will need a very different amount of HCl to reach the target pH. If your recipe calls for Tris base and you only have Tris-HCl, you will need approximately 31.5 g of Tris-HCl to get the same molar amount as 24.2 g of Tris base, and you will need far less additional HCl for pH adjustment because the hydrochloride salt already contributes acid.
Overshooting the pH is another common stumble. Concentrated HCl is powerful, and a few extra drops can push you well below the target. If you do overshoot, you can add small amounts of Tris base powder back, but this alters your concentration. A better approach is to slow down as you approach the target, adding HCl drop by drop and waiting several seconds between additions for the meter to stabilize. Some people switch to a dilute HCl solution (1 M) for the final adjustment to reduce the risk of overshooting.
Using tap water instead of distilled or deionized water is a mistake that may not produce an immediately obvious problem but can cause subtle issues downstream. Tap water contains variable amounts of minerals, chlorine, and dissolved gases that can interfere with sensitive assays, introduce unwanted ions, or promote microbial growth in stored solutions.
Not calibrating your pH meter before use is a silent source of error. A meter that is off by even 0.2 pH units means your buffer is off by the same amount. Calibrate with at least two standard buffers (pH 4.0 and pH 7.0, or pH 7.0 and pH 10.0) before every session.
Scaling Up or Down
The recipe above is for 1 liter of 10x stock. If you routinely go through large volumes of working TBS, it can make sense to prepare 2 or even 4 liters of stock at once. Simply multiply all quantities by the scaling factor. For 2 liters: 48.4 g Tris base, 160 g NaCl, adjusted to pH 7.6 and brought to 2 liters total volume.
Scaling down works the same way. For 500 mL of 10x stock, use 12.1 g of Tris base and 40 g of NaCl. The only thing that changes is the vessel size. pH adjustment is done the same way regardless of volume, though smaller volumes require proportionally less HCl, so be even more cautious about overshooting.
Some labs prepare 20x stocks to save shelf space. This is fine in principle, but solubility becomes a consideration. At 20x concentration, the NaCl content reaches about 2.74 M, which approaches saturation at room temperature (NaCl saturates around 5.3 M at 20 °C, so you still have margin). The real concern is that a 20x stock diluted to 1x introduces more room for pipetting error than a 10x stock, since you are working with smaller volumes of concentrate. For most purposes, 10x strikes the right balance between shelf space and ease of use.
Water Quality and Dissolved Gases
The quality of water you use for buffer preparation has a surprisingly large effect on performance, especially for assays that are sensitive to trace metal contamination or ionic interference. Deionized water (Type II or better) is the minimum standard for buffer preparation. For particularly sensitive work like mass spectrometry-compatible buffers or nucleic acid applications, Type I ultrapure water is worth the extra effort.
Dissolved COâ‚‚ is worth thinking about even in freshly dispensed water. Distilled and deionized water in equilibrium with the atmosphere has a pH around 5.5 due to dissolved carbon dioxide forming carbonic acid. When you add this water to your Tris solution, the carbonic acid is neutralized by the buffer, which is exactly what a buffer is designed to do. But if you are preparing a large batch and the water has been sitting in an open carboy absorbing COâ‚‚ for days, the extra acid load can consume more of your buffering capacity than expected. Using freshly dispensed water and keeping containers capped avoids this issue.
Glassware cleanliness also plays a role that is easy to overlook. Residual detergent from washing, or traces of other buffers and reagents from previous use, can alter pH or introduce contaminants. Rinse all glassware with distilled water immediately before use, and dedicate specific containers to buffer storage if possible.