TL;DR: For a common immunoblotting formulation, 1X TBS contains 20 mM Tris and 150 mM NaCl at pH 7.4–7.6. TBST is TBS plus Tween 20, commonly 0.05–0.1% v/v. A 10X TBS stock can be prepared at 200 mM Tris and 1.5 M NaCl, then diluted 1:10.
Tris-buffered saline (TBS) is one of the most widely used laboratory wash and antibody-dilution buffers in Western blotting, immunohistochemistry and immunofluorescence. Adding a non-ionic detergent produces TBST, which improves removal of weakly bound proteins and antibodies from membranes.
There is no single universal TBS formulation. Published protocols vary in Tris concentration, sodium chloride concentration and pH. This guide uses a practical immunoblotting formulation of 20 mM Tris, 150 mM NaCl, pH 7.6 for 1X TBS, with 0.1% Tween 20 for TBST. If your antibody or assay has been validated with a different formulation, keep the validated formulation.
1X TBS composition
| Component | Final concentration |
|---|---|
| Tris | 20 mM |
| NaCl | 150 mM |
| pH | 7.4–7.6 |
10X TBS recipe
| Final volume | Tris base | NaCl |
|---|---|---|
| 100 mL | 2.42 g | 8.77 g |
| 500 mL | 12.11 g | 43.83 g |
| 1 L | 24.23 g | 87.66 g |
How to make 1 L of 10X TBS
- Add approximately 700–800 mL purified water to a clean beaker.
- Dissolve 24.23 g Tris base and 87.66 g NaCl with gentle stirring.
- Measure the pH at room temperature.
- Adjust to pH 7.6 gradually with HCl.
- Bring to a final volume of 1 L with purified water.
- Store in a clean, labelled bottle. Sterile-filter if the intended use requires sterility.
How to make 1X TBS from 10X stock
Dilute the stock 1:10. For 1 L, mix 100 mL 10X TBS with 900 mL purified water.
How to make TBST
For Western blot washing, a common starting concentration is 0.1% Tween 20. Some antibodies and low-background workflows perform better at 0.05%.
| Final volume | 10X TBS | Tween 20 for 0.1% | Water |
|---|---|---|---|
| 100 mL | 10 mL | 100 µL | to 100 mL |
| 500 mL | 50 mL | 500 µL | to 500 mL |
| 1 L | 100 mL | 1 mL | to 1 L |
Why Tween 20 changes Western blot performance
Tween 20 is a non-ionic surfactant. In wash buffer it can reduce weak hydrophobic interactions and improve removal of non-specifically bound antibody. Too little detergent may increase background; too much can reduce weak but genuine antibody-antigen interactions. The optimum therefore depends on the antibody, membrane and blocking chemistry.
TBS vs PBS for Western blotting
Both buffers are widely used. TBS is often preferred for phospho-protein work because phosphate-containing buffers can interfere with some phosphatase-related workflows or selected antibody systems. PBS can be perfectly suitable when already validated. The key is to avoid changing wash-buffer chemistry halfway through a validated experiment without checking background and signal.
Common mistakes
- Adjusting Tris pH while the solution is warm: Tris pH is temperature-dependent. Measure near the temperature at which the buffer will be used.
- Adding viscous Tween 20 inaccurately: pre-wet the pipette tip, pipette slowly and allow full aspiration/dispensing.
- Assuming 0.1% is mandatory: 0.05% may reduce signal loss for some antibodies.
- Reusing contaminated wash buffer: discard cloudy or visibly contaminated buffer.
Troubleshooting
| Problem | Likely cause | What to try |
|---|---|---|
| High background | Insufficient washing or detergent too low | Increase wash number/duration or test 0.1% Tween 20. |
| Weak signal | Over-stringent washing | Reduce Tween 20 to 0.05% or shorten washes. |
| Inconsistent pH | Temperature differences during preparation | Adjust pH at room temperature and record temperature. |
Frequently asked questions
What does TBST stand for?
Tris-buffered saline with Tween 20.
Can I make TBST directly from 10X TBS?
Yes. Dilute 10X TBS to 1X and add Tween 20 to the required final concentration.
Can I autoclave TBST?
It is usually better to sterilise the TBS stock first, then add detergent afterward if sterility is required. Heat can affect some surfactant formulations.
Related ABMIUM guides
Primary research and methods references
- Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. PNAS. 1979.
- Burnette WN. “Western blotting”: electrophoretic transfer of proteins and antibody detection. Anal Biochem. 1981.
- Long-term storage of diluted antibody in Tris-buffered saline with Tween 20. 2025.