TBST, or Tris-Buffered Saline with Tween 20, is a widely used wash buffer in molecular biology, primarily for techniques like Western blotting. It maintains a stable environment for biological samples while effectively removing unbound materials.
Key Components and Necessary Equipment
Preparing TBST requires specific chemical components. Tris base provides buffering capacity, maintaining a stable pH. Sodium chloride (NaCl) contributes to the buffer’s tonicity. Tween 20, a non-ionic detergent, reduces non-specific binding of antibodies or probes. These components are combined with deionized or distilled water, which serves as the solvent.
Several pieces of laboratory equipment are essential for accurate TBST preparation. A precision balance weighs solid components. A pH meter adjusts the solution’s pH, typically to 7.6. A magnetic stirring plate with a stir bar ensures thorough mixing. Graduated cylinders measure liquid volumes, and glassware like beakers and reagent bottles are needed for preparation and storage.
Step-by-Step Preparation Guide
To prepare 1 liter of 1X TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), accurately weigh the solid components. Measure 2.42 grams of Tris base and 8.77 grams of sodium chloride. These measurements ensure the correct buffer concentration.
Transfer the weighed Tris base and sodium chloride into a clean 1-liter beaker. Add approximately 800 milliliters of deionized or distilled water to the beaker. Place the beaker on a magnetic stirring plate with a stir bar, allowing the solids to dissolve completely while stirring gently. Ensure all the powder has gone into solution before proceeding.
Once the solids are fully dissolved, carefully measure 1.0 milliliter of Tween 20 using a serological pipette or a small graduated cylinder. Add the Tween 20 to the solution in the beaker while it continues to stir. The detergent will mix into the solution, preparing it for the next critical step.
The next step involves adjusting the pH of the solution. Using a calibrated pH meter, monitor the pH of the stirring solution. Slowly add hydrochloric acid (HCl), typically a 1 M or 6 M solution, drop by drop, until the pH reaches 7.6. It is important to add the acid gradually, allowing the pH to stabilize between additions, as overshooting the target pH requires adding more Tris base to correct.
After the pH is adjusted, carefully transfer the solution to a 1-liter graduated cylinder. Add additional deionized or distilled water to bring the total volume precisely to 1000 milliliters (1 liter). This final volume adjustment ensures the correct concentration of all components. Pour the finished TBST into a clean storage bottle.
Storage and Practical Considerations
Proper storage of prepared TBST is important for maintaining its stability and effectiveness over time. Store the buffer at room temperature (approximately 20-25°C) in a clean, airtight reagent bottle. Storing it in a cool, dark place can help prolong its shelf life, though refrigeration is not typically necessary unless specified for particular applications.
Under these conditions, a properly prepared 1X TBST solution can generally be stored for several weeks to a few months without significant degradation. Always label the storage bottle clearly with the buffer’s name, concentration, the date of preparation, and the initials of the person who prepared it. This practice helps track the buffer’s age and ensures proper laboratory management.
To extend the utility of the buffer and save preparation time, many laboratories opt to prepare a concentrated stock solution, such as 10X TBST. This concentrated stock can be stored for longer periods and diluted with deionized water to 1X concentration just before use. Diluting a stock solution reduces the risk of contamination in the working solution and ensures its freshness for experiments.