How to prepare Phosphate Buffered Saline (PBS)?

Phosphate buffered saline (PBS) is a buffer solution extensively used in biological research for immunohistochemistry (IHC) screening. PBS is a water-based salt solution including sodium hydrogen phosphate, sodium chloride, and, in certain situations, potassium chloride as well as potassium dihydrogen phosphate.

How to prepare Phosphate Buffered Saline (PBS)
How to prepare Phosphate Buffered Saline (PBS)

Phosphate buffered saline (a.k.a. PBS or PBS buffer) is one of the most often used buffers since it is isotonic to most cells. PBS’s ion concentrations and osmolarity match those of the human body, making this solution isotonic. Because it is harmless to cells, it is often used for cell container rinsing and other preparations that may leave a residue. It is easy to make and has a long shelf life, however it will precipitate in the presence of zinc ions.

Apparatus Required for the Preparation of 1X PBS

To prepare a 1X (the standard concentration for research) solution of phosphate buffered saline (PBS), you will need the following materials:

  • 1-L graduated cylinder
  • 2-L beaker
  • Stir bar
  • Stir plate
  • pH meter
  • Autoclave
  • Glass bottle for storage

To prepare 1 L of PBS, combine the following ingredients in the following amounts:

ComponentQuantity
Distilled water800 mL
Sodium Chloride (NaCl)8 g
KCl (potassium chloride)0.2 g
Na2HPO4 (disodium phosphate)1.44 g
KH2PO4 (monopotassium phosphate)0.24 g
Dilute HCl and/or NaOHSmall amount to final pH adjustment

How to Prepare 1X PBS

  • When making a solution, you want to keep it moving. This will help things disintegrate quicker. To avoid splashing while adding water, place the stir bar in the empty beaker and then on top of the stir plate.
  • A stir bar has a magnet that allows it to spin and agitate the solution when a container of liquid is put on the stir plate, which has a motor.
  • Before turning it on, wait until there is a solution in the beaker.
  • Start with a less amount of water when making a solution than you will need. The remaining water will be added at the end to complete the 1 L total.
  • Measure 800 mL of water into the graduated cylinder, then pour the entire amount into a clean beaker.
  • The stir plate should be set at a medium speed with the water still in the beaker. As the stir bar starts to spin, you will see a little whirlpool forming in the water.
  • Weigh 8 grams of NaCl, 0.2 grams of KCl, 1.44 grams of Na2HPO4, and 0.24 grams of KH2PO4. ¬†Measure all the chemicals separately using the balance. If you accidentally add too much of a chemical, just take it out until you have the proper amount.
  • While the water is swirling, add each chemical. You can add them all at once because there is no specific sequence in which they should be included. Allow the mixture to stir continuously until it has completely dissolved.
  • After everything has completely dissolved, you must change the buffer’s pH until it reaches 7.4. Place the pH meter into the top of the solution while keeping the stir bar in place. Allow the pH meter’s figures to cease fluctuating. You may probably need to add sodium hydroxide (NaOH) to the solution to alter the pH because it is probably excessively acidic.
  • Bring your solution up to the final volume of 1 liter once the pH has been suitably adjusted. In the 1-L graduated cylinder, carefully pour the whole contents of the beaker. To create 1 L, add enough distilled water.
  • Place the lid on the bottle, but do not screw it down. When autoclaving, the pressure in the bottle might build up and burst if the lid is tightened. Put a piece of autoclave tape over the lid to keep it in place while autoclaving.
  • Set the autoclave to sanitize at 15 psi for 20 minutes on the liquid cycle.

Apparatus Required for the Preparation of 10X PBS

Preparing the 10X (ten times the regular concentration used) stock is nearly comparable to creating the 1X stock; simply add 10 times the amount of ingredients. You will need the following supplies in order for making a 10X stock:

  • 1-L graduated cylinder,
  • Weighing balance,
  • 2-L beaker,
  • Stir bar,
  • Stir plate,
  • pH meter,
  • Autoclave, and
  • Glass bottle for storage.

To manufacture one liter, you will need the following quantities of each compound:

ComponentQuantity
Distilled water700 mL
NaCl (sodium chloride)80 g
KCl (potassium chloride)2 g
Na2HPO4 (disodium phosphate)14.4 g
KH2PO4 (monopotassium phosphate)2.4 g
Dilute HCl and/or NaOHSmall amount to final pH adjustment
The final concentrations of each substance in the 10X solution are 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4.

Uses of Phosphate Buffered Saline (PBS)

Because it is isotonic and safe for most cells, phosphate-buffering saline has a wide range of applications.

  • It may be used to dilute things and is frequently employed to rinse cells out of containers.
  • Due to the way the water molecules in PBS are organized, it may be utilized as a dilutent in a variety of ways to dry biomolecules. Take proteins as an example. It will be “dried” and rendered immobile on a stable surface.
  • To stop the cells from being destroyed, the pH is kept constant and stable.
  • The thin layer of water that binds to the material inhibits denaturation and other conformational changes. Carbonate buffers can be used for the same purpose, although they are less effective.
  • PBS may also be used as a reference spectrum for evaluating protein adsorption in ellipsometry.

Additional Tips

  • Always attempt to prepare the buffer at the temperature you want to use it at, as temperature variations might cause a dissociation shift.
  • Ensure that the pH meters are properly maintained.
  • To eliminate the danger of contamination, work in aseptic circumstances. After preparing the buffer, sterilize it to avoid microbial growth, which is prevalent in phosphate-containing and neutral pH buffers like 1x PBS.
  • If you make a 10x PBS solution instead of a 1x PBS solution, you can dilute 100ml of the 10X solution in 900ml of distilled water to get 1l of 1x PBS buffer.

Video on How to Prepare Phosphate Buffered Saline

YouTube video

References

  • https://stjohnslabs.com/how-to-prepare-phosphate-buffered-saline-pbs/
  • https://www.aatbio.com/resources/buffer-preparations-and-recipes/pbs-phosphate-buffered-saline
  • https://www.sigmaaldrich.com/NP/en/support/calculators-and-apps/1x-phosphate-buffered-saline
  • https://www.wikihow.com/Make-Phosphate-Buffered-Saline
  • https://static.igem.org/mediawiki/2019/9/90/T–IISER_Kolkata–cell_6.pdf
  • https://sharebiology.com/phosphate-buffered-saline-pbs-composition-and-preparation/
  • https://chemistry.stackexchange.com/questions/112025/how-can-i-prepare-a-pbs-phosphate-buffer-solution-ph-7-4
  • https://www.thoughtco.com/make-phosphate-buffered-saline-375492

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Jyoti Bashyal is an enthusiastic researcher currently working in the field of computational chemistry. She is a graduate student from the Central Department of Chemistry, Tribhuvan University. As a writer, she relishes creative writing and believes in hard work with a positive mindset.

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