Phosphate Buffered Saline Protocol (PBS) and Use


Phosphate Buffered Saline protocol (PBS) is  used in biological applications, such as washing cells, transportation of tissues, and dilutions. Phosphate buffer are widely used because they help maintain a constant pH level in a particular environment and has minimum PH change in 4-23 C°. It is nontoxic to cells and isotonic; so, it is used for cell container rinsing and other preparations that might leave a residue. It is simple to prepare and has good shelf life.

PBS can autoclave or filter for sterilization which is perfect for a general use buffer. But it has some downsides in compatibility with other materials. Divalent Iones such as Ca, Mg, Zn, Fe, Cu, and others, can make constant precipitations with PBS. Also, at 4C°, PBS can precipitate by itself. In this case, precipitates can easily resolve by increasing temperature to 37C°.

In ELISA and other immunoassay tests, PBS significantly increases test recovery and has negative Matrix effect on most protein and peptides and all lipids. Even as a washing buffer, PBS can is not proper buffer unless that be used with especial additives. If PBS is used as HRP- Conjugated Buffer in ELISA it can significantly reduce Ag-Ab complex formation and leave unacceptable changes in Indirect ELISA tests Threshold, Gray-Zone, and sensitivity. Fortunately, by adding adjusting chemicals, we still use it as a effective conjugate buffer, reaction buffer and Wash Buffer in ELISA.


Phosphate Buffered Saline preparation Protocol

PBS can be made as a 1× solution or as a 10× stock.

    • Dissolve Following listed chemicals in 850 ml Distilled water.

               Item                             amount 1X PBS                                amount 10X PBS

             NaCl                                        8gr                                                        80gr

              KCl                                         0.2gr                                                       2gr

             Na2HPO4                              1.44gr                                                   14.1gr

             KH2PO4                                 0.24gr                                                      2.4gr

      • Adjust the pH to 7.4 value by slowly adding concentrated HCl, if you need.
      • Adjust the volume of the solution to 1000 mL with distilled water.
      • If necessary, Filter Tris buffer with 0.22 μm sterile filter. Also, PBS cab be autoclaved for sterilization.

Note1: For Immunoassay tests, you do not need to sterilize.

Note2: Phosphate Buffered Saline Protocol (PBS) is stable at 4C° at least for 2 weeks.

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