Tris-Buffered Saline (TBS) is a fundamental solution in biological and biochemical research. It is a balanced mixture that provides a stable chemical environment for various biological materials and processes. Preparing a 10x TBS stock solution is standard practice in laboratories. This concentrated stock saves time, reduces preparation frequency, and ensures consistency across experiments.
Essential Components and Concentrations for 10x TBS
A standard 10x TBS solution uses two primary components: Tris base and sodium chloride. Tris(hydroxymethyl)aminomethane (Tris base) is the buffering agent that resists pH changes. Its conjugate acid has a pKa of approximately 8.07, stabilizing the solution in the biologically relevant pH range of 7.4 to 8.0.
Sodium chloride (NaCl) provides the correct ionic strength. This salt concentration mimics the physiological environment, making the solution isotonic and preventing damage to biological samples. The 10x stock solution is typically prepared to contain 200 mM Tris and 1,500 mM (or 1.5 M) NaCl.
Calculating Reagent Weights
Accurate preparation requires precisely calculating the mass of each chemical needed for the final volume. The calculation uses the formula: Mass (grams) = Molarity (moles/Liter) \(\times\) Molar Mass (grams/mole) \(\times\) Volume (Liters). For a 1 L batch of 10x stock, the molar mass for Tris base is \(121.14 \text{ g/mol}\).
To achieve \(200 \text{ mM}\) (\(0.20 \text{ M}\)) Tris in 1 L, the calculation is \(0.20 \text{ M} \times 121.14 \text{ g/mol} \times 1.0 \text{ L}\), resulting in \(24.228 \text{ grams}\) of Tris base. Sodium chloride (molar mass \(58.44 \text{ g/mol}\)) must be weighed separately. The required mass of NaCl for a \(1.5 \text{ M}\) concentration in 1 L is \(1.5 \text{ M} \times 58.44 \text{ g/mol} \times 1.0 \text{ L}\), which equals \(87.66 \text{ grams}\).
These masses (\(24.228 \text{ g}\) Tris base and \(87.66 \text{ g}\) NaCl) are necessary for a 1 L batch of 10x stock. Using precise, analytical balances ensures the final solution has the correct buffering capacity and ionic strength.
Step-by-Step Preparation Protocol
Preparation begins by dissolving the calculated masses of Tris base and sodium chloride in high-quality deionized water. Use approximately 900 milliliters of water for a 1 Liter final volume, leaving room for pH adjustment. Add the solid chemicals to a flask and stir continuously using a magnetic stirrer until completely dissolved.
The next step is pH adjustment, which is a sensitive stage of the preparation. Use a calibrated pH meter to monitor the solution while slowly adding concentrated hydrochloric acid (HCl). The target pH for standard 10x TBS is typically \(7.6\) at room temperature.
Adding the acid establishes the buffering equilibrium by converting Tris base into its conjugate acid form. Add the acid slowly, drop by drop, waiting for the pH reading to stabilize after each addition. Adding too much acid will require the use of a base to correct it, unnecessarily increasing the salt concentration. Once the pH is confirmed at \(7.6\), transfer the solution to a volumetric flask. Bring the final volume up to the 1 L mark by adding the remaining deionized water.
Storage and Dilution of 10x TBS Stock
The concentrated 10x Tris-Buffered Saline stock must be stored correctly to maintain its stability. Transfer the solution to an airtight container and label it clearly with the name, concentration, pH, and preparation date. The stock can be stored at room temperature or refrigerated at \(4^\circ \text{C}\).
The 10x stock typically remains stable and usable for up to three months. When a working solution is needed, the 10x stock must be diluted to its final 1x concentration using a simple ten-fold dilution. This involves combining one part of the 10x stock with nine parts of deionized water.
For example, making 1 Liter of the 1x working solution requires mixing 100 milliliters of the 10x stock with 900 milliliters of water. This dilution procedure yields the final \(50 \text{ mM}\) Tris, \(150 \text{ mM}\) NaCl solution at \(\text{pH } 7.6\) for biological experiments.