Protocol: HEPES Buffer Recipe
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
sterilize.
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.
