Buffers and Lab Consumables

When it comes to obtaining optimal results in your research, having the right tools and reagents makes all the difference. One critical aspect of experimental design is choosing the right buffering system that provides solution stability and pH control without interfering with the explored biological processes or reactions. Even slight changes in pH can lead to adverse molecular interactions (i.e., protein-protein and protein-ligand), protein unfolding, and functional inactivities that significantly impact the success and reproducibility of an experiment.

To simplify your research needs with accurate and reliable results, AAT Bioquest offers several buffers for general lab use in cell culture, cytology, bioconjugation, molecular biology, and proteomics, as well as a comprehensive portfolio of cell culture media formulations and buffer recipes. In addition, we provide a full range of general laboratory supplies and consumables needed to achieve your goals for a variety of applications. Whether in need of plasticware, basic lab equipment, reagents, and buffers, AAT Bioquest has the solution to support your routine lab work.

What Is a Buffer?

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Buffers are organic substances that resist rapid and significant changes in pH levels, provide solution stability, and supply essential salts and nutrients for cells and tissues. To effectively maintain a specific pH range, buffers consist of a weak acid (proton donor, HA) and its conjugate base (proton acceptor, A^-), or vice versa. This mixture allows the buffering solution to neutralize the effects of any added hydrogen ions (H⁺) or hydroxide ions (OH^-) so that equilibrium of the system can be maintained. The balanced equation for this reaction is:

HA ⇌ H⁺ + A^-

According to Le Chatelier's principle, when a strong acid is added to a buffer (e.g., more H⁺), the equilibrium shifts to the left. The conjugate base reacts with hydrogen ions from the strong acid, thereby increasing the concentration of the weak acid. Similarly, if a strong base is added, the equilibrium shifts to the right. The weak acid releases hydrogen ions which react with hydroxide ions to form water and the weaker conjugate base of the acid. These two reactions can continue to alternate back and forth, such that the pH remains stable within a very narrow range.

The degree to which pH is sustained upon adding acids or bases to a buffer system is referred to as the 'buffer capacity' and is typically defined by the acid's dissociation constant, or pK_a (-logK_a). Buffer capacity generally depends upon the concentration of the buffer solution. Buffers with higher concentrations offer higher buffering capacity. It is important to note that most buffers function optimally when the pK_a of the conjugate weak acid used is close to the desired working range of the buffer, at least within one pH unit of the target pH.

 

Considerations for a 'Good' Buffer

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Buffers are selected based on the experiment that will be performed. Since most biochemical processes function effectively under physiological conditions, buffers with a pH range of 6.0 to 8.5 are generally preferred. However, pH is not the only criteria to consider when choosing a buffer. By 1980, Good and his colleagues identified several additional parameters likely to be of value in life science research. These include:

• A pK_a between 6.0 and 8.0 - the optimal pH for most biological reactions falls between this range.
• High water solubility - biological processes favor aqueous environments; therefore, buffers should exhibit good solubility in water. Likewise, minimum solubility in nonpolar solvents prevents buffer compounds from accumulating in nonpolar compartments (e.g., cell membranes).
• Inertness - buffers must not influence, participate, or interfere with any biological processes, reactions, or components. For instance, when labeling proteins, such as antibodies, with amine-reactive dye succinimidyl esters (e.g., iFluor^® 488 succinimidyl ester), avoid using buffers containing amines. Primary amine buffers like Tris are not compatible and will compete with the conjugation reaction.
• Minimal salt effects - buffer components should not interact or affect ions involved in the biochemical reactions being studied.
• Known complex-forming tendency with metal ions - complex formation between ions and buffer components releases protons, which affects the pH of the system and may compromise results. Thus, complexes should remain soluble, and their binding constant must be known. For enzyme assays (e.g., PCR), metal complexation can be an issue since many enzymes need metal ions to function properly. For these applications, choose a buffer with a low metal-binding constant. If your experimental design requires using a metal, then choose a buffer that does not form a complex with that metal.
• Non-toxic to cells - buffers must not kill your sample
• No interference with cell membranes - buffers must not penetrate the cell membrane and be allowed within the cytosol. Zwitterionic buffers, such as MOPS and HEPES, are membrane-impermeant.
• Very low optical absorbance - buffers should not absorb light at wavelengths > 230 nm to prevent interferences in spectrophotometric assays.
• Minimally affected by changes in temperature, concentration, and ionic strength - an ideal buffer is one whose buffering capabilities (pK_a) are not affected by the concentration, temperature, and ionic composition of the medium. A good rule of thumb is to make the buffer at the temperature you plan to use it.
• Chemical stability - buffers should not degrade under working conditions, oxidize, or be affected by the system it is being used in.
• Convenient and cost-effective - buffer preparation and purification should be easy and inexpensive.

Most of the buffers used in cell cultures, isolation of cells, enzyme assays, immunoassays, and other biological applications satisfy the aforementioned characteristics laid out by Good and company. For instructions on how to make a specific cell culture media or buffer solution, refer to our cell culture media or buffer recipes databases.

Cell Lysis Buffers

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Effective cell lysis buffers are essential in molecular biology experiments for breaking down cell membranes and compartments and facilitating the extraction of target macromolecules (e.g., proteins and nucleic acid species). Lysis buffers typically contain buffering and ionic salts to regulate the pH and osmolarity of the lysate, inhibitors to preserve the integrity of the target molecules, and one or more detergents to lyse and solubilize membrane structures.

Lysis Buffers for Protein Extraction

Several criteria are used to determine the type of lysis buffer required for an experiment. These include the type and cell source, the desired molecule or structure, and the level of their functionality. For protein extraction, lysis buffers typically contain a cocktail of protease and phosphatase inhibitors and either an ionic, non-ionic, or zwitterionic detergent (see Table 2 below).

 

Ionic detergents can be either anionic or cationic, such as sodium dodecyl sulfate (SDS) or ethyl trimethyl ammonium bromide. They are considered the harshest, totally disrupting membranes and denaturing proteins by breaking protein-protein interactions. Ionic detergents are well-suited for gel electrophoresis (e.g., SDS-PAGE) or any other application that involves modifying proteins and disrupting cellular structures. Non-ionic detergents, such as Triton X-100, NP-40, and Tween 20, are milder in comparison to ionic detergents. These non-denaturing detergents break protein-lipid and lipid-lipid interactions rather than protein-protein interactions. They are essential for isolating and purifying enzymes or multimeric proteins in their native state. Zwitterionic detergents exhibit characteristics of both ionic and non-ionic detergents. They have an overall neutral net charge and are effective at breaking protein-protein interactions while maintaining the native state and charge of the individual proteins. Zwitterionic detergents are used in chromatography, 2D gel electrophoresis, mass spectrometry, and the solubilization of organelles and inclusion bodies.

Lysis Buffers for Nucleic Acid Extraction

Compared to protein extraction, nucleic acid extraction is much simpler. Since nucleic acids are far more resilient to denaturation than most proteins, the lysis buffer will commonly contain denaturing detergents. For RNA extraction, harsh denaturing agents, such as guanidine thiocyanate, phenol, and chloroform, and RNase inhibitors, are usually added to the lysis buffer. The guanidinium thiocyanate-phenol-chloroform method disrupts the cells, denatures the proteins, and deactivates the nucleases to stabilize the DNA, RNA, and protein. Centrifugation then separates the sample into an upper aqueous phase containing the RNA and a lower organic phase containing DNA and protein. Total RNA can then be recovered by precipitation with isopropanol. For DNA extraction, lysis buffers typically contain SDS and, depending upon the type of DNA (e.g., genomic, mitochondrial, or plasmid ), can include other additives. For example, lysis buffers for extracting plasmid DNA will contain sodium hydroxide for alkaline lysis and potassium acetate for renaturation of the plasmid DNA.

AAT Bioquest offers a range of optimized reagents designed to effectively lyse cells and extract proteins and nucleic acids from various starting materials and sample sizes, including bacterial, mammalian, viral, and plant cultures. Our ReadiUse™ lysis buffers and ReadiPrep™ cell fractionation kits are formulated to obtain high protein, DNA, or RNA yields from tissues, cells, or subcellular fractions, with more consistent results and minimal hands-on time. The purified proteins, DNA, or RNA can then be used in a wide range of downstream applications such as enzyme assays, chromatographic analysis, electrophoresis, PCR, and RT-PCR.

Cell Lysis Buffers	Cell Fractionation and Organelle Specific Lysis Buffers	Detergents	Resources
ReadiUse™ Bacterial Cell Lysis Buffer ReadiUse™ Mammalian Cell Lysis Buffer ReadiUse™ Viral RNA Lysis Buffer ReadiUse™ Yeast Cell Lysis Buffer	ReadiPrep™ Mitochondrial/Cytoplasmic Fractionation Kit ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit	ReadiUse™ 10% Triton X-100	Biological Detergent Properties and Applications Buffer Preparations and Recipes Properties of Common Protease and Phosphatase Inhibitors Common Enzymes/Proteins and Their Inhibitors Genomic DNA Extraction Protocol

CytoWatch™ Series

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The CytoWatch™ series is made up of solutions ranging from blocking reagents to buffers for various instrumentation platforms to aid in imaging. CytoWatch™ materials are either ready-to-use or only require simple dilution. This is for the convenience of the researcher as well as easier extended storage. For example, the CytoWatch™ Wash-free fluorescence cell imaging buffer *10X* is a ready-to-use buffer optimized for fluorescence cell imaging. In some cases, this buffer significantly enhances the imaging signal. It is used in wash steps when performing immunohistochemistry (IHC) or immuno-labeling with tissue or 3D cell culture. The buffer is 10X concentrated and should be diluted to 1X with PBS before use.

 

Laboratory Consumables

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AAT Bioquest's consumables and general laboratory equipment include, microcentrifuge tubes, single channel pipettes and pipette tubes, reservoirs, spin filters, spin columns and spin adapters. All of our laboratory consumables are made of high quality materials and could be useful in a number of laboratory fields and applications.

Additional Resources

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Cell Lysis Detergents

Detergents are critical components in cell lysis buffers for their ability to solubilize membrane lipids and proteins. The amount of detergent needed for optimal protein extraction depends on the CMC, aggregation number, temperature, and nature of the membrane and the detergent. The solubilization buffer should contain sufficient detergent to provide greater than one micelle per membrane protein molecule to help ensure that individual protein molecules are isolated in separate micelles. Detergents may need to be removed downstream if they interfere with analysis or production.

Protease and Phosphatase Inhibitors

Immediately following lysis, activities such as proteolysis, dephosphorylation and denaturation begin consequently degrading proteins of interest and their activation states. These events, which are carried out by proteases and phosphatases, can be slowed down significantly if samples are kept on ice or at 4°C and appropriate inhibitors are added to the lysis buffer.

Ordering Information

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