Many biological experiments require the use of buffers to maintain an effective pH, which is important since proteins and enzymes are sensitive to changes in pH, and choosing the right buffer for current and downstream experiments is critical. In this article, I will briefly discuss some important factors to take into consideration when choosing your buffer and then discuss how to prepare the most commonly used buffers in life science.

Choosing A Buffer

When choosing a buffer, it is important to take into consideration each buffer’s advantages and disadvantages. First and foremost, the pH range of the agents you plan to use in your experiment needs to coincide with the pH range of the buffer you choose. You can determine if they coincide by looking at the pH range of the buffer or by actually comparing the pKa of your buffer to the pH you intend to keep your experiment at. The pKa value should be within one unit of your desired pH.

Additionally, you must take into consideration concentration, buffer toxicity, temperature, and reactivity. The concentration of your buffer must be sufficient to account for the amount of acid or base you plan to use in your experiment. The more of the pH-altering component that you plan to use, the higher the buffer concentration you will need. Buffer toxicity is important because some buffers can be toxic to the cells you are working with, so if you are unsure about toxicity you can test the buffer out on your cells before you use it in your experiment. You should also ensure that the buffer you decide to use is not capable of producing unwanted reactions in your experiment. Last, but not least, check that the temperature you plan to use in your experiment works with your buffer of choice because temperature can alter the buffering capacity of your buffer.

For more information on choosing the right buffer for your experiment, refer to our user guide or our short article to help determine what is most appropriate.

learn to prepare your biological buffers with this helpful handbook. The download is free. it includes information about choosing a buffer, protocols, intro to buffers, charts and more.



How to Make Bis-Tris Stock Solution

Bis-Tris is a member of the Bis(2-hydroxyethyl)amine family of buffers. You can use this buffer to resist pH changes in experiments where pressure is being varied, in gel electrophoresis experiments, and during anion exchange chromatography and NMR. The pH range of this buffer is 5.8 to 7.2.

Bis-Tris Preparation Steps:

To prepare 1 liter of 1M Bis-Tris buffer stock solution, dissolve 209.24 g of GoldBio Bis-Tris in 750 mL of dH2O. Adjust to desired pH using concentrated hydrochloric acid. A table is available for you to use in the Bis-Tris PDF protocol. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store buffer at 4 degrees Celsius.

Things to Watch Out for:

When working with a Bis-Tris buffer solution, remember that Bis-Tris forms complexes with lead and copper and various other metals. In addition to the uses mentioned above, Bis-Tris can be substituted as a safer alternative for cacodylate, which is a toxic buffering agent.



How to Make HEPES Stock Solution

HEPES is a zwitterionic Good’s buffer effective for a pH range of 6.8 to 8.2. This makes HEPES an effective buffer at physiologic pH. This buffer can be used in cell culture media for a variety of organisms. It can also be used as a binding buffer in protein studies, used in cation exchange elution experiments, and in gel electrophoresis as a running buffer.

HEPES Preparation Steps:

To prepare 1 liter of 1M HEPES buffer solution, dissolve 238.30 g of GoldBio HEPES in 750 mL of dH2O. Adjust to desired pH using 10N sodium hydroxide. A table is available for you to use in the 1M HEPES PDF protocol. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store buffer at 4 ˚C. If you are working with GoldBio HEPES Sodium Salt, use the HEPES-Na protocol instead.

Things to Watch Out for:

HEPES is soluble in water, inexpensive, and is typically biologically inert. However, it does participate in oxidation-reduction reactions yielding free radicals and interferes with reactions between DNA and restriction enzymes, but to a lesser extent than Tris due to steric hindrance. It is also important to note that HEPES should not be used when working with heteromeric connexin channels as it can inhibit their function. If you are working with toxicity studies, choose HEPES Sodium Salt over the free acid.



How to Make MES Stock Solution

MES is a buffer of the morpholinic family. Like HEPES, MES is a zwitterionic Good’s buffer. This buffer is used in solutions that contain metal ions because it does not form complexes with most metals. MES is typically used in buffered culture media for yeast, bacteria and mammalian cells, but it is toxic to plants at concentrations higher than 10 mM. MES is also a great choice of buffer for a variety of chromatography and electrophoresis experiments because it displays low ionic mobility and conductivity. It is an effective buffer in pH ranges from 5.5 to 6.7.

MES Preparation Steps:

To prepare 1 liter of 0.5M MES free acid buffer solution, suspend 97.62 g of GoldBio MES free acid in 750 mL of dH2O. Adjust to the desired pH using 10N sodium hydroxide. A table is available for you to use in the 0.5M MES PDF protocol. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store at 4 ˚C. If you are working with GoldBio MES MonohydrateorMES Sodium Salt, use the MES Monohydrate protocolorMES-Na protocol instead.

In addition to its typical uses, MES is a great alternative to cacodylate, citrate, or malate that is non-toxic.



How to Make PIPES Stock Solution

PIPES is a member of the piperazinic family of buffers, the same family as HEPES. It is a zwitterionic Good’s buffer that is effective for a pH range of 6.1 to 7.5. With a pH range slightly different than that of HEPES, PIPES is effective at half the concentration of HEPES. This buffer does not complex with metals and is therefore useful in solutions containing metal ions. It can also be used for chromatography, fixation of plant cells for electron microscopy, and as a replacement for cacodylate buffer.

PIPES Preparation Steps:

To prepare 1 liter of 1M PIPES free acid buffer solution, add 302.37 g of GoldBio PIPES free acid to 600 mL of dH2O. Adjust to desired pH using 10N sodium hydroxide. A table is available for you to use in the 1M PIPES PDF protocol. Fill to a final volume of 1L with dH2O and sterilize using filter or autoclave. Store at 4 ˚C. If you are working with GoldBio PIPES sodium salt, use the 1M PIPES-Na PDF protocol instead.

Things to Watch Out for:

PIPES free acid will not readily dissolve until the solution is raised above pH 6.5, however PIPES sodium salt is readily soluble. If you are working with the free acid form of the buffer, use sodium hydroxide to convert it to a sodium salt for increased solubility at a lower pH. Like HEPES, PIPES is not suitable for experiments involving oxidation reduction reactions because it can lead to formation of free radicals.



How to Make TES Stock Solution

TES is a buffer that is a structural analog to Tris buffer. It is also a zwitterionic Good’s buffer. It is effective for a pH range of 6.8 to 8.2. TES is a unique buffer because its pKa coincides exactly with physiologic pH (7.4) at 25 ˚C. This makes TES a useful biological buffer in a variety of applications such as precipitation and extraction of DNA, gel filtration, and chromatography.

TES Preparation Steps:

To prepare 1 lister of 1M TES buffer, dissolve 229.25 g of GoldBio TES in 750 mL of dH2O. Adjust to desired pH using 10N sodium hydroxide. A table is available for you to use in the 1M TES PDF protocol. Fill to a final volume of 1L with dH2O and sterilize with filter or autoclave. Store at 4 ˚C.

TES is can be used in TEST-yolk buffer medium. This buffer is used in the refrigeration and transport of semen.



How to Make Tris and Tris HCl Stock Solutions

Tris or (hydroxymethyl) aminomethane is used in a variety of buffer solutions including TAE and TBE. Tris can also be used for running and loading buffers, such as for SDS-PAGE. Its effective pH range is 7.0 to 9.0. Two common forms of the Tris buffer are Tris base and Tris HCl. Adjusting the pH of Tris base can be challenging, but using Tris HCl rather than adjusting pH with NaOH or HCl addition can simplify this process. Other advantages to using Tris HCl as an alternative to NaOH or HCl include safety and reproducibility. More about Tris versus Tris HCl has been discussed in GoldBio’s article This vs. That.

Tris Base & Tris HCl Preparation Steps:

Tris: To prepare 1 liter of 1M Tris buffer, dissolve 121.14 g of GoldBio Tris in 750 mL of dH2O. Adjust to desired pH using concentrated hydrochloric acid. A table is available for you to use in the 1M Tris PDF protocol. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store at 4 ˚C.

Tris HCl: To prepare 1 liter of 1M Tris HCl buffer, dissolve 157.60 g of GoldBio Tris HCl in 750 mL of dH2O. Adjust to desired pH using 10N sodium hydroxide. A table is available for you to use in the 1M Tris HCl Buffer protocol. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store at 4 ˚C.

Things to Watch Out for:

One tip to using Tris solutions is that the pH of the solution is extremely dependent on temperature. For this reason, Tris should be prepared at the temperature at which the buffer will be used. Additionally, you should avoid using single-junction pH electrodes containing silver with a Tris buffer.



How to Make TAE and TBE Buffers

TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are both used for gel electrophoresis. They are typically used in procedures for the separation of nucleic acids. TBE has a better buffering capacity than TAE. TAE and TBE each have unique advantages, and information about choosing between these two buffers can be found just below the preparation steps.

TAE & TBE Preparation Steps:

TAE: To make a 50X buffer solution of TAE, combine 242.28 g of GoldBio Tris with 18.61 g GoldBio EDTA disodium. Add 750 mL of dH2O and dissolve. Add 57.1 mL glacial acetic acid and fill to a volume of 1 L with dH2O. Store at room temperature. The TAE PDF protocol is also available. A 1:50 dilution of TAE stock solution with dH2O will make a 1X working solution with a pH of about 8.6.

TBE: To make a 10X stock solution of TBE, combine 108 g of GoldBio Tris with 55 g of GoldBio Boric Acid. Add 750 mL of dH2O and dissolve. Add 40 mL 0.5M (7.5 g) EDTA Disodium and fill to a final volume of 1 L with dH2O. Store at room temperature. A 1:10 dilution of TBE stock solution with dH2O will make a 1X working solution with a pH of about 8.3.

Things to Watch Out for:

When both TAE or TBE could work for your experiment, choosing between them can be a little tricky. TAE can be used to speed up the process as linear, double-stranded DNA runs quickly in TAE than TBE. It is also important to note that the boric acid in TBE can inhibit many enzymes, so TAE should be chosen if downstream steps rely on enzymatic processes.

One thing to note if you are working with TBE, it should be diluted as mentioned above to 1X or even to 0.5X due to higher concentrations causing generation of large amounts of heat that can alter you experiment.



How to Make TE Buffer

TE (Tris-EDTA) is a buffer commonly used to solubilize DNA or RNA. It works well because it protects these nucleic acids from degradation.

TE Preparation Steps:

To prepare 1 liter of 10X TE buffer, combine 100 mL of 1M Tris buffer as prepared above with 20 mL of 0.5M EDTA disodium. Fill to a final volume of 1L with dH2O and sterilize by filter or autoclave. Store at room temperature. A 10X TE PDF protocol is available for your use. A 1:10 dilution of TE stock solution with dH2O will create a 1X working solution containing 10mM Tris and 1mM EDTA.

Things to Watch Out for:

Typically, TE is used at a certain pH based on whether you are working with DNA or RNA. It can be adjusted to pH 7.5 for RNA experiments and pH 8.0 for DNA experiments. Both DNA and RNA can be stored using TE buffer of pH 8.0.



How to Make PBS

PBS (Phosphate Buffered Saline) is an isotonic, nontoxic buffer used to mimic the physiologic pH, osmolarity, and ion concentrations of humans. It is typically used in tissue culture at pH 7.4 and for immunoassays such as Western blots and ELISA assays. PBS can also be used to dilute protein samples, purify protein samples, and as a wash buffer.

GoldBio PBS buffer tablets make a solution that contains 10mM phosphate buffer, 137mM sodium chloride, and 2.7mM potassium chloride.

Things to Watch Out for:

When working with PBS, chelation of calcium and magnesium ions can lead to adhesion and clumping in solution because phosphate can form precipitates with these ions. EDTA can be used to disengage attached or clumped cells, but use of PBS should be reserved for experiments that do not require use of PCR in later steps.



Using this article, you can prepare the most commonly used buffers in life science. For additional tips and protocols on using buffers, check out GoldBio’s User Guide for Buffers. It has troubleshooting tips as well as frequently asked questions for the buffers discussed here.



protocols for tris, hepes, aces, ada, bis-tris, chaps, boric acid and more all found in this free biological buffers handbook. Download here





Rebecca Talley
GoldBio Staff Writer


Rebecca is a medical student at the University of Missouri.
She previously worked as a lab technician while studying
biology at Truman State University. As an aspiring
reproductive endocrinologist with an interest in global
health, Rebecca has traveled across Central America on
medical mission trips. With a passion for the life sciences,
she enjoys writing for GoldBio.


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