how to make PBS Buffer?

A PBS buffer solution is a handy buffer to have around, especially for biological applications, but how to make PBS Buffer? The goal of a buffer solution is to help maintain a stable pH when a small amount of acid or base is introduced into a solution.

Phosphate-buffered saline (PBS) is an isotonic solution that is used in many biological research applications. To make 1 L of PBS, add 100 mL of 10X PBS to 900 mL of water. This PBS recipe contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4.

PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface.

It is applied in washing cells after protein expression procedures due to its osmolarity and pH to avoid cell disruption to preserve and subsequently freeze the cells for downstream molecular biology applications. 

how to make PBS Buffer

Material Required For Preparation of Phosphate Buffer Saline (PBS)

  • Sodium phosphate monobasic (anhydrous) (NaH2PO4)
  • potassium dihydrogen phosphate (KH2PO4)
  • Sodium chloride (NaCl)
  • Potassium chloride (KCL)
  • Phosphoric acid (H2SO4) makes the pH more acidic and sodium hydroxide (NaOH) makes the pH more alkaline
  • Scale and weigh boats
  • Magnetic stirrer and stir bar
  • A pH probe that’s calibrated and appropriate solutions for adjusting pH
  • 1L volumetric flask
  • Tween 20 (optional)

Recipe for Making PBS Buffer

You can prepare PBS in several ways. There are multiple formulas. Some of them don’t contain potassium, while others contain calcium or magnesium. 

This recipe is relatively easy. It’s for 10X PBS stock solution (0.1M). However, you can also make a 1X stock solution, or begin with this 10X recipe and dilute it to 1X. The whole process takes about 10 minutes and an option to add Tween is also provided.

A typical chemical composition of 1X PBS has a final concentration of 10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl. Here’s the final concentration of reagents in the solution:

SaltConcentration (mmol/L)Concentration (g/L)
NaCl1378.0
KCl2.70.2
Na2HPO4101.42
KH2PO41.80.24

Prepare the Phosphate Buffer

  • For Making 1L of PBS Buffer solution star with 800 ml of Distilled water to dissolve salts in an appropriate container.
  • Use an electronic balance to accurately weigh out all salt powders
  • Add 8 g of Sodium chloride NaCl to the solution.
  • Add 0.2 g of Potassium Chloride (KCL) to the solution.
  • Add 1.44 g of Sodium Phosphate Dibasic (NaH2PO4) to the solution.
  • Add 0.245 g of Potassium Phosphate Monobasic (KH2PO4) to the solution.
  • Adjust the solution to desired pH (typically pH ≈ 7.4) with the help of NaOH and HCL
  • After dissolving all substances make the total volume of solution 1 liter with distilled water.

The pH of PBS is ~7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid (HCl) or sodium hydroxide (NaOH).

PBS can also be prepared by using commercially made PBS buffer tablets or pouches.

If used in cell culture, the solution can be dispensed into aliquots and sterilized by autoclaving or filtration. Sterilization may not be necessary depending on its use.

PBS can be stored at room temperature or in the refrigerator. However, concentrated stock solutions may precipitate when cooled and should be kept at room temperature until the precipitate has completely dissolved before use.

Tips for Making PBS Buffer

Store the buffer at room temperature after you’ve made the PBS solution.

Non-anhydrous reagents can be substituted but you’ll have to recalculate the appropriate mass of each to accommodate the added water molecules.

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