Buffer Recipes

Buffer Recipes


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Description: HEPES is a general-purpose zwitterionic buffer which does not bind magnesium, calcium, manganese(II) or copper (II) ions. Buffer strength for cell culture applications is usually in the range of 10 to 25 mM. After the addition of HEPES, the pH is adjusted with NaOH or HCl. Care must be taken to maintain the appropriate osmolality in media, and toxicity with respect to a given cell line must be evaluated. HEPES is reportedly superior to sodium bicarbonate in controlling the pH in

tissue and organ cultures. HEPES may exhibit toxicity at concentrations greater than 40 mM. Studies have indicated that 20 mM HEPES is the most satisfactory concentration of the buffer when both Hanks' and Earle's solutions are used. CO2 incubators should not be used with media buffered solely with HEPES. HEPES is not recommended for certain protein applications such as the Folin-Ciocalteu protein assay; however, it does not affect the Biuret protein assay. 
HEPES is the buffer of choice in a protein deposition technique in electron microscopy because it did not affect metal substrates.
HEPES has been evaluated and shown to be suitable for use with Ampholines in generating pH gradients less than 1 pH unit wide for isolectric focusing applications.
HEPES is the recommended buffer for the glutamate binding assay because it prevents binding to non-receptor materials.
A buffer solution of HEPES can be prepared by any of several methods. The free acid can be added to water, then titrated with approximately one-half mole equivalent of sodium hydroxide or potassium hydroxide to the desired pH. A simple mixing table for preparing 0.05 M HEPES/NaOH has been published. Alternatively, equimolar concentrations of HEPES and of HEPES sodium salt
can be mixed in approximately equal volumes and back-titrate with either solution to the appropriate pH. The sodium salt can be titrated with HCl to yield a half-equivalent of sodium chloride; however, the addition of the ionic strength will change the osmolality of the solution. Most solutions of HEPES hemisodium will disolve in water forming a pH at 7.5 with little to no adjustment.
Typical Formulations for HEPES buffered Saline (2X):
Formula #1:
Dissolve above ingredients in a total volume of 90 ml of distilled water. Adjust the pH to 7.05 with 0.5 N NaOH, and then adjust the
volume to 100 ml with distilled water. Sterilize the solution by passage through a 0.22 micron filter. Store in aliquots at -20°C.
Formula #2:
Add the above ingredients and stir until dissolved. Titrate to pH 7.05 with 5 M NaOH (an exact pH is extremely important). Filter
Test the formulation before use with transfection experiments. Test by mixing 0.5 ml of the above buffer with 0.5 ml 250 mM CaCl2 and vortexing. A fine precipitate should develop that is readily visible in the microscope. If this precipitate does not form, do not use the batch of buffer for transfection experiments.
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Tris Solution

Tris is used to make a basic buffering solution. The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1°C. Consequently, tris-buffered solutions should be adjusted to the desired pH at the temperature at which they will be used. Because the pKa of Tris is 8.08, avoid using Tris as a buffer below pH 7.2 or above pH 9.0.

To prepare a 1M stock solution of Tris-Cl:

  • Dissolve 121 g Tris base in 800 ml H2O
  • Adjust to desired pH with concentrated HCl. Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and 42 ml for a pH 8.0 solution
  • Adjust volume to 1 liter with H2O
  • Filter sterilize if necessary
  • Store up to 6 months at 4°C or room temperature
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PBS Recipe/phosphate buffered solution

Phosphate buffered saline (abbreviated as PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

Phosphate buffered saline Applications:

PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. 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. The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers may be used for the same purpose but with less effectiveness. PBS can be used to take a reference spectrum when measuring the protein adsorption in ellipsometry.
Additives can be used to add function. For example, PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good’s buffers are recommended.

10X PBS Recipe

There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium. One of the most common preparations is described below.
A 10 liter stock of 10x PBS can be prepared by dissolving
  • 800 g NaCl,
  • 20 g KCl,
  • 144 g Na2HPO4 · 2H2O
  • 24 g KH2PO4
  • 8 L of distilled water.
After complete mixing, top up final solution to 10 L. The pH of the 10X stock is will be approximately 6.8, but when diluted to 1x PBS it should change to 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 or sodium hydroxide.
On dilution, the resultant 1x PBS should have a final concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and a pH of 7.4.
Another preparation is described in Molecular Cloning by Sambrook, Fritsch and Maniatis, Apendix B.12 as follows:

1 liter of 1X PBS:

  1. Start with 800 ml of distilled water:
  2. Add 8 g of NaCl.
  3. Add 0.2 g of KCl.
  4. Add 1.44 g of Na2HPO4.
  5. Add 0.24 g of KH2PO4.
  6. Adjust the pH to 7.4 with HCl.
  7. Add distilled water to a total volume of 1 liter.
Dispense the solution into aliquots and sterilize by autoclaving (20 min, 121°C, liquid cycle). Store at room temperature.
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SDS Page Gel (Preparing SDS-Page Gels)

An anionic detergent typically used to solubilize and denature 
proteins for electrophoresis. SDS has also been used in large-scale phenol extraction of RNA to promote the dissociation of protein from nucleic acids when extracting from biological material. Most proteins bind SDS in a ratio of 1.4 grams SDS to 1 gram protein. The charges intrinsic to the protein become insignificant compared to the overall negative charge provided by the bound SDS. The charge to mass ratio is essentially the same for each protein and will migrate in the gel based only on protein size.

Typical Working Concentration: > 10 mg SDS/mg protein

Typical Buffer Compositions:

SDS Electrophoresis Gel Running Buffer:

Composition (g/L)
Molarity or %
0.025 M
0.192 M
pH 8.3

SDS Electrophoresis Gel Sample Solubilization Buffer

Composition (g/L)
Molarity or %
0.125 M
Titrated to pH 8.0 with HCl
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