A.1.2 Preparation of phosphate buffer solution. Preparation of phosphate buffer solution Preparation of phosphate buffer solution

Phosphate-saline wash buffer with Tween-20 for immunohistochemistry is a concentrate (20x), which, after dilution, is used to wash slides of reagents and short-term storage of immunohistochemical samples between procedures. After dilution, the ready-to-use 0.01 M solution has a pH of 7.4 ± 0.1. The use of this phosphate-buffered saline allows not only to ensure high-quality washing, but also to preserve the morphological characteristics of the antibodies used and their epitopes, which facilitates the specific binding necessary for the immunohistochemical reaction. Adding Tween-20 to phosphate-buffered saline promotes more efficient washing and prevents nonspecific staining.

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Application

Sodium phosphate buffer is widely used because it is isotonic and nontoxic to cells. PBS is used to dissolve substances to rinse containers containing cells.

Preparation

There are many ways to prepare sodium phosphate buffer. Some formulas contain no potassium, others contain calcium or magnesium.

To prepare 1 liter of one-time sodium phosphate buffer, use:

• 8.00 g NaCl
• 0.20 g KCl
• 1.44 g Na 2 HPO 4
• 0.24 g KH 2 PO 4
• dissolved in 800 ml distilled water
• adjust pH to 7.4 with hydrochloric acid or sodium hydroxide
• add distilled water to 1 liter.

Ten liters of tenfold PBS stock solution can be prepared by dissolving 800 g NaCl, 20 g KCl, 144 g Na 2 HPO 4 and 24 g KH 2 PO 4 in eight liters of distilled water, bringing the volume up to ten liters. The pH of the resulting solution will be approximately 6.8, but after dilution to 1X PBS will be 7.4. After preparing the solution, check the pH value using a pH meter. If necessary, you can adjust the pH using hydrochloric acid or sodium hydroxide.

The simplest way to prepare sodium phosphate buffer is to use commercially available tablet preparations. Such tablets are diluted with distilled water to a given volume and a solution of a given concentration is obtained.

For cell cultures, the solution should be aliquoted and sterilized by autoclaving (20 minutes at 121°C, liquid mode). Sterilization is not necessary, depending on the application. It is possible to store sodium phosphate buffer at room temperature, but to prevent the growth of bacteria, long-term storage of non-sterile buffer is recommended in the refrigerator. Salts in concentrated stock solutions may precipitate when cooled, so before use it is recommended to warm the concentrated solution to room temperature and wait until the precipitate is completely dissolved.

Notes

see also

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2010.

Phosphate Buffered Saline: 1 L of 10X

Description

PBS: 1 L of 10X (Phosphate buffered saline) is a concentrated buffer solution ready for dilution to a 1X PBS working solution commonly used in biological research. PBS buffer is ideal for maintaining a constant pH, and since it is isotonic and non-toxic to cells, it can be used in a wide variety of biological applications. PBS is frequently used as a wash and dilution buffer, and is also used to take a reference spectrum when measuring protein adsorption.

Usage
Prior to use, dilute to a 1X solution and filter through a 0.22 µm filter into a sterile flask. Makes 10 L of 1X PBS buffer with a phosphate buffer concentration of 0.01M, 0.027M potassium chloride, 0.137M sodium chloride, and 1.76 mM potassium phosphate. The PBS solution pH will be approximately 7.4.
. If buffer is used in cell culture applications, sterilization of the solution may be necessary. Pre-diluted buffer can be dispensed into aliquots and sterilized by autoclaving (20 min, 121°C, liquid cycle).

. PBS can be stored at room temperature, but non-sterile solutions may warrant refrigeration to prevent bacterial growth over time. Concentrated stock solutions may precipitate when cooled and should be kept at room temperature until precipitate has completely dissolved before use.

A buffer solution commonly used in biological research

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Name: PBS buffer (phosphate buffered saline), 10X, 1 L / Phosphate Buffered Saline: 1 L of 10X. Note: Species specificity: . Description: . Application: . Delivery on blue ice, storage at room temperature,

Chemical grade reagents are used to prepare buffer solutions. and analytical grade, specially prepared. The preparation of reagents is carried out as follows.

Potassium phosphate monosubstituted, KH 2 PO 4 , molecular weight 136.09. 100 g of the drug is dissolved when heated to boiling in 150 ml of water. The solution is filtered while hot. With constant stirring, the filtrate is cooled to 10 ºС. Then add 150 ml of ethyl alcohol. The crystals released during constant stirring of the filtrate are filtered off using a suction funnel and recrystallized again under the same conditions; the crystals are dried to constant weight at 105…110 ºС. If there is a drug with a content of the main substance in the range of 99.9...100.0%, no preliminary preparation of the substance is carried out.

Disubstituted sodium phosphate, Na 2 HPO 4 · 12H 2 O, molecular weight 358.12. There are two ways to prepare the drug.

a) 150 g of the drug is dissolved in 150 ml of water when heated to 100 0 C. The solution is filtered hot and after cooling, the precipitated crystals are filtered out. Recrystallization is repeated by heating to 100 ºС. The recrystallized drug is heated in a porcelain cup in a water bath with continuous stirring until the drug is completely dry. The resulting salt is dried in a desiccator over fused calcium chloride for 24 hours. The content of the main substance in the recrystallized preparation (Na 2 HPO 4 ·2H 2 O) is checked. To do this, about 0.5000 g of the drug is dissolved in 50 ml of water, 2...3 ml of a saturated sodium chloride solution is added and titrated with 0.1 N hydrochloric acid solution in the presence of a methyl red indicator. If necessary, make adjustments to the sample size. 1 ml of exactly 0.1 N hydrochloric acid solution corresponds to 0.0178 g of Na 2 HPO 4 2H 2 O.

b) 75 g of the drug is dissolved in 250 ml of water heated to 60 ºС. The solution is filtered while hot, the filtrate is cooled with constant stirring to 10 ºС. The precipitated crystals are filtered off using a suction funnel and recrystallized again under the same conditions. The resulting salt is first dried at a temperature not exceeding 30 ºС for 24 hours, then continued to dry in an oven at 50 ºС for 3...4 hours, and finally at 120±5 ºС to a constant weight, preventing the salt from melting. After drying, the salt has the composition Na 2 HPO 4.

After preparing the reagents, prepare initial solutions of monosubstituted potassium phosphate and dibasic sodium phosphate.

A sample of anhydrous potassium phosphate monosubstituted KH 2 PO 4 weighing 9.078 g is dissolved in water and the volume of the solution is adjusted to 1 liter. To stabilize the solution, add 3...4 drops of toluene.

A sample of sodium phosphate disubstituted Na 2 HPO 4 ·12H 2 O, weighing 11.876 g, is dissolved in water and the volume of the solution is adjusted to 1 liter. To stabilize the solution, add 3...4 drops of toluene.

Phosphate buffer solutions with pH from 4.94 to 9.18 are prepared from the initial solutions in accordance with Table A.2.

Table A.2 - Phosphate buffer solution with pH 4.94...9.18

Date added: 2015-08-06 | Views: 4058 |

PBS (phosphate buffered saline) is a commonly used buffer for immunohistochemical staining and is commonly used in biological research. The concentration of osmolarity and ions of solutions corresponds to the concentration of the human body (isotonic).

PBS is a water-based solution containing sodium hydrogen phosphate, sodium chloride and, in some cases, potassium chloride and potassium dihydrogen phosphate.

Immunofluorescence staining for IHC

Immunofluorescence staining was the first immunohistochemical (IHC) staining method. With the basis of the antigen-antibody binding reaction, antigens become visible using fluorescent dyes when conjugated with antibodies. The process occurs when activated by excitation light of a specific wavelength under a fluorescence microscope. Immunohistochemistry refers to the process of detecting antigens (such as proteins) in cells of a tissue section using the principle of antibodies that specifically bind to antigens in biological tissues.

Using Phosphate Buffered Saline (PBS)

Phosphate buffered saline has many uses because it is isotonic and nontoxic to most cells. It can be used to dilute substances and is used to rinse containers containing cells. PBS can be used as a diluent in biomolecule drying methods because the water molecules within it will be structured around the substance (such as a protein) to "dry" it and immobilize it to a solid surface.

A thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers can be used for the same purpose, but with less efficiency. PBS can also be used to provide a reference spectrum when measuring protein adsorption in ellipsometry.

Preparation of PBS

There are many different ways to prepare PBS.

Some formulas contain no potassium, while others contain calcium or magnesium. The buffer recipe below, which is relatively simple, is for a 10X stock solution of PBS (0.1M). It is also possible to add Tween. The whole process takes about 10 minutes.

How to Make Phosphate Buffered Saline (PBS)

1. Weigh the following: 10. 9 g sodium phosphate anhydrous (Na2HPO4), 3.2 g sodium phosphate anhydrous (NaH2PO4) and 90 g sodium chloride (NaCl).
2. Dissolve all of the above in only 1 liter of distilled water.
3. Adjust the pH value to 7. 4.
4. Make the solution to a final volume of 1 liter.
5. (not necessary). For a solution containing 0.5% Tween 20, add 5 ml of Tween 20 to the 1L solution.
6. Dilute 10X before use and adjust pH if necessary.

• Store at room temperature.
• Non-femoral reactants can be substituted, but you will have to recalculate the appropriate mass of each to accommodate the added water molecules.

What do you need to create a PBS buffer?

• Single phase sodium phosphate (anhydrous)
• Dibasic sodium phosphate (anhydrous)
• Sodium chloride
• Scale and weight boats
• Magnetic stirrer and stirrer > pH probe, calibrated and suitable solutions for pH adjustment
• 1 L volumetric flask
• Tween 20 (optional)