TBST is one of the most straightforward buffers you will ever make in a lab, and getting it right comes down to three ingredients: Tris base, sodium chloride, and Tween 20. A standard formulation uses 10 mM Tris and 150 mM NaCl adjusted to around pH 7.6, with 0.1% Tween 20 (v/v) stirred in at the end. Despite this simplicity, small errors in pH, detergent concentration, or storage can quietly wreck a Western blot, so understanding why each component is there matters as much as weighing the powders.
The Standard Recipe
Most Western blotting protocols call for a 1× working solution of TBST at the bench. A widely used formulation dissolves Tris base and NaCl in deionized or distilled water, adjusts the pH, and then adds Tween 20. For one liter of TBST at working strength, the components are:
- Tris base: 1.21 g (giving a final concentration of 10 mM)
- NaCl: 8.77 g (giving a final concentration of 150 mM)
- Tween 20: 1 mL (giving a final concentration of 0.1% v/v)
- Water: deionized or distilled, to a final volume of 1 liter
Dissolve the Tris base and NaCl in roughly 900 mL of water on a magnetic stirrer. Once both are fully in solution, adjust the pH to 7.6 using concentrated hydrochloric acid, then bring the volume to one liter. Add Tween 20 last and stir gently to mix it without generating excessive foam. This order matters because pH adjustment is easier before the detergent goes in, and frothing a detergent-containing solution introduces bubbles that are annoying to work with and can interfere with membrane washing steps.
Making a Concentrated Stock
Preparing a 10× TBS stock and diluting it on the day of use saves time when you run blots frequently. For one liter of 10× TBS, dissolve 12.1 g of Tris base and 87.7 g of NaCl in about 800 mL of water, adjust the pH to 7.6, and bring the volume to one liter. When you are ready to blot, dilute 100 mL of 10× TBS into 900 mL of water and add 1 mL of Tween 20. Keep the Tween 20 out of the stock concentrate. Its job is to reduce background during antibody incubation and washing, and leaving it in a concentrated stock for weeks encourages microbial growth and oxidation that can degrade the detergent over time.
A 10× stock stored at room temperature in a tightly capped bottle is stable for months. Label it clearly as TBS without the “T” so no one accidentally uses the concentrate for washing. The diluted 1× TBST should ideally be prepared fresh or used within a week. You will not see it go cloudy or change color when it spoils, so dating the bottle is the easiest safety net.
Why These Three Components
Each ingredient in TBST plays a specific role during Western blotting, and understanding those roles helps you troubleshoot when results look off.
Tris keeps the pH stable. After you transfer proteins to a membrane, every subsequent step involves soaking or incubating that membrane in solution. Antibodies bind their targets within a narrow pH window, roughly 7.2 to 7.6 for most immunodetection protocols. Tris acts as a buffering agent that resists pH drift during the hours a blot spends in wash and incubation steps. Using 10 mM Tris is enough buffering capacity for a system that does not generate significant acid or base during the procedure.
Sodium chloride controls ionic strength. At 150 mM, it approximates the salt concentration of physiological fluids, which keeps antibody-antigen interactions behaving as they would under normal biological conditions. Too little salt can increase non-specific binding because charged proteins stick to the membrane electrostatically. Too much salt can weaken specific antibody-antigen interactions and reduce your signal.
Tween 20 is a mild non-ionic detergent that reduces background noise on the blot. During washing steps, it displaces proteins that are sitting on the membrane surface through weak, non-specific interactions while leaving tightly bound antibody-antigen complexes intact. The typical concentration is 0.1%, though some protocols use 0.05%.
1PubMed Central. Long-term Storage of Diluted Antibody in Tris-buffered Saline with Tween 20 – Section: MethodsGetting the pH Right
Adjusting to pH 7.6 is straightforward but worth doing carefully. Dissolve the Tris and NaCl first, let the solution equilibrate to room temperature, and then add concentrated HCl dropwise while monitoring the pH with a calibrated meter. Tris dissolved in water typically starts at a pH above 10, so it takes a noticeable amount of acid to bring it down. Approach the target gradually because overshooting below 7.4 means you either need to add more Tris base (which changes the molarity) or start over.
Temperature affects Tris pH more than you might expect. Tris has an unusually large temperature coefficient: its pH drops by about 0.03 units per degree Celsius increase. A solution adjusted to pH 7.6 at room temperature will read noticeably differently if you check it at 4 °C. For Western blotting this rarely causes trouble because incubation and washing happen at room temperature or 37 °C, but if you calibrate the pH in a cold room and then use the buffer at the bench, the actual working pH will be lower than what you measured. Adjust at the temperature you plan to use the buffer.
How Much Tween 20 Matters More Than You Think
Many protocols treat the Tween 20 concentration as interchangeable between 0.05% and 0.1%, but the difference is not trivial. The detergent’s job is to strip weakly bound proteins from the membrane without disturbing the antibody-antigen complexes you want to detect. Research has shown that even at 0.05%, Tween 20 can dissociate some proteins from nitrocellulose, and the degree of stripping depends on how tightly each individual protein binds to the membrane surface.2Journal of Immunological Methods. Tween 20 removes antibodies and other proteins from nitrocellulose Some proteins hang on and barely budge. Others are nearly completely removed by the detergent.
This means that cranking up the Tween 20 concentration to fight high background can backfire. If your target protein binds loosely to the membrane, a higher detergent concentration may strip it right along with the non-specific noise. When you have persistent high background, it is tempting to throw more detergent at the problem. A better first step is to increase the number or duration of washes with your current TBST rather than changing the Tween 20 percentage. Three washes of ten minutes each at 0.1% Tween 20 is a common starting point, and many labs go to five washes before touching the detergent concentration.
TBST Versus PBST
You will see some protocols call for PBST (phosphate-buffered saline with Tween 20) instead of TBST. The two are functionally similar for most Western blot applications, but there are situations where the choice matters. If your detection system uses alkaline phosphatase-conjugated secondary antibodies, PBST is a poor choice because the phosphate ions in PBS competitively inhibit the enzyme. Alkaline phosphatase needs to hydrolyze a phosphate-containing substrate to generate signal, and free phosphate in the buffer competes for the active site. TBST does not have this problem, which is one reason it became the default in many labs.
For HRP (horseradish peroxidase)-based detection, either buffer works. If your lab runs blots with both enzyme systems, standardizing on TBST avoids the risk of someone accidentally using the wrong wash buffer on an alkaline phosphatase blot. This is one of those practical considerations that saves more headaches than any optimization experiment.
Common Preparation Mistakes
A few errors come up repeatedly in labs, especially among newer researchers preparing the buffer for the first time.
Pipetting Tween 20 is awkward. The liquid is extremely viscous and clings to pipette tips, making accurate volume measurement difficult. Cutting the tip of a 1-mL pipette tip to widen the bore helps, or you can weigh the Tween 20 instead (its density is close to 1.1 g/mL, so 1 mL weighs about 1.1 g). Some researchers dip the pipette tip into the buffer and let the solution rinse the Tween 20 off, which works but requires patience.
Using Tween 20 that has oxidized will produce poor results. Tween 20 degrades over time, forming peroxides. Old, yellowed bottles of Tween 20 are a known source of increased background and erratic signals. If the bottle has been open for more than a year or looks discolored, replace it. Storing Tween 20 in small aliquots or in bottles with minimal headspace slows oxidation.
Forgetting to re-check pH after topping up the volume is another common slip. Adding water to bring the solution to its final volume dilutes the acid slightly, which can nudge the pH up. The shift is usually small, but if you are working right at the edge of 7.6, it can push you to 7.7 or higher. A quick re-check after volume adjustment takes seconds and avoids uncertainty.
Storage and Shelf Life
Concentrated 10× TBS stores well at room temperature for several months. The diluted 1× TBST is less stable, primarily because the dilute Tween 20 in an aqueous solution supports microbial growth over time. Some labs store working TBST at 4 °C to slow this, though room temperature is fine for a buffer you will use up within a week. If you notice any turbidity or floating particulates, discard the buffer and make a fresh batch. Adding sodium azide at 0.02% to stored TBST can prevent microbial contamination, but sodium azide inhibits HRP, so any buffer preserved this way must never come into contact with your blot during the detection step. Most labs find it simpler to just make fresh TBST regularly.
One group investigated the long-term storage of diluted antibody solutions in TBST, confirming that the buffer itself supports stable antibody performance over extended storage periods when handled properly.3PubMed Central. Long-term Storage of Diluted Antibody in Tris-buffered Saline with Tween 20 This suggests that contamination and detergent degradation, rather than any inherent instability of the buffer system, are the main enemies of shelf life.
When the Standard Formulation Is Not Enough
Some Western blotting applications call for tweaks to the basic TBST recipe. If you are working with fluorescent detection rather than chemiluminescence, the autofluorescence of Tween 20 becomes a consideration. Research on fluorescent Western blotting found that Tween 20’s autofluorescence is negligible when the membrane is imaged wet but can become significant when the detergent dries on the blot surface.4ScienceDirect. Optimization of blocking conditions for fluorescent Western blot If you image dry membranes with a fluorescence scanner, thorough final washes with plain TBS (no Tween 20) before drying can prevent this artifact.
The same study noted that adding a small amount of sodium dodecyl sulfate (SDS) at 0.005% to 0.02% can reduce background caused by non-specific antibody binding. The trade-off is that SDS, being a harsher ionic detergent, also weakens specific antibody-antigen interactions, reducing your signal along with the noise. This is a knob you turn only when gentler approaches have failed and you are willing to accept some signal loss in exchange for a cleaner background.
Scaling for High-Throughput or Large Experiments
Researchers running many blots in a week often prepare several liters of TBST at once. The recipe scales linearly, so doubling or tripling all components and water volume works without adjustment. For very large batches, a carboy with a spigot makes dispensing easier and reduces contamination from repeatedly opening a bottle. Use a stir plate large enough to handle the volume, and check that the Tris and NaCl are fully dissolved before adjusting pH. Undissolved Tris at the bottom of a carboy will slowly release, drifting the pH upward over days.
Some labs prepare large volumes of 20× TBS to save space. This is fine as long as you confirm that all solutes remain in solution at the higher concentration. NaCl at 3 M (the equivalent of 20× the standard 150 mM) approaches its solubility limit at room temperature, so warming the water slightly during preparation helps. Let it cool before checking pH, and make sure no crystals form during room-temperature storage.
TBST in Other Blotting Applications
Although this article focuses on Western blotting, TBST shows up in other immunodetection protocols. Dot blots, slot blots, and many ELISA-like membrane assays use TBST for the same reasons: it provides a stable pH environment, physiological ionic strength, and gentle detergent action for washing steps. The recipe is identical in most cases, though ELISA plate-washing protocols sometimes use lower Tween 20 concentrations (0.05%) because the plastic surface of a microplate has different protein-binding characteristics than a nitrocellulose or PVDF membrane. If you move from blotting to plate-based assays, check the assay kit’s recommendations before assuming your standard 0.1% TBST is appropriate.
For immunohistochemistry and immunofluorescence on tissue sections, some protocols substitute Triton X-100 for Tween 20 because Triton permeabilizes cell membranes more aggressively, allowing antibodies to reach intracellular targets. Tween 20 is milder and better suited to surface-level detection, which is exactly what membrane-based blotting requires. Mixing the two up is a mistake that occasionally happens when a lab runs both types of experiments and keeps multiple wash buffers on the shelf. Labeling bottles clearly and keeping blotting buffers physically separated from tissue-staining buffers prevents this.