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?
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-
Acid and base added to buffer.
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 pKa
). 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 pKa
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
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 pKa 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 (pKa) 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
Table 1. Commonly used biological buffers.
|ACES||6.1-7.5||6.88||6.78||6.54||182.2||Zwitterionic||Cell culture media, protein purification, enzymatic assay (glucosidase activity), x-ray crystallography, yeast, and bacterial studies||Strong: Cu and Mg|
Weak: Ca, Mn, Co, Ni, and Zn
|ADA||6.0-7.2||6.65||6.59||6.46||190.155||Zwitterionic||Protein crystallization, electrophoresis, differential scanning calorimetry, and metal decontamination in soil.||Strong: Mn, Co, Ni, Zn, Cd, Pb, and Cu|
|BES||6.4-7.8||7.17||7.09||6.90||213.2||Zwitterionic||Cell culture media, chromatography, protein quantification, transfection, bacterial endotoxin studies||Strong: Cu|
|Bicine||7.6-9.0||8.35||8.26||8.04||163.2||Zwitterionic||Cell culture media, chromatography, PCR, protein separation||Strong: Fe, Co, Mg, Ca, Ni, Zn, and Cd|
|Bis-Tris||5.8-7.2||n/a||6.50||6.36||209.2||Zwitterionic||Cell culture media, chromatography, electrophoresis, protein purification||Strong: Cu and Pb|
Weak: Ca, Cd, Co, Mg, Mn, Ni, and Zn
|CAPS||9.7-11.1||10.56||10.40||10.02||221.32||Zwitterionic||Cell culture media, chromatography, electrophoresis, electroblotting, diffusion blotting||Negligible|
|CHES||8.6-10.0||9.55||9.49||9.36||207.3||Zwitterionic||Cell culture media, chromatography, electron paramagnetic resonance (EPR) spectroscopy, electrophoresis, enzymatic assays, kinetic measurements, yeast dextrose medium||Weak: Cu, Pb, Cd, and Zn|
|HEPES||6.8-8.2||7.55||7.45-7.65||7.31||238.3||Zwitterionic||Cell culture media, chromatography, cryopreservation, electrophoresis, electroporation, protein quantification||Negligible|
|HEPPSO||7.1-8.5||n/a||7.80||6.66||268.33||Zwitterionic||Cell culture media, enzymatic studies, isoelectric focusing, protein quantification, toxicology studies||Weak: Cu|
|MES||5.5-6.7||6.16||6.10||5.97||195.2||Zwitterionic||Cell culture media, chromatography, electrochromatography, electrophoresis, fluorescence microscopy, toxicology studies in yeast||Strong: Fe|
Weak: Cu, Mg, Mn, and Ni
Cell Lysis Buffers
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
Table 2. Common Mammalian Cell Lysis Buffers for General Protein Extraction
|Purpose||Recommended for the extraction of cytoplasmic, nuclear, membrane-bound and mitochondrial proteins. It contains harsh denaturing, ionic detergents and milder non-ionic detergets. Disrupts protein-protein interactions.||Recommended for extraction of cytoplasmic and membrane-bound proteins. It is a milder, non-ionic detergent. Proteins retain their native state, and protein-protein interactions are preserved.|
- 25 mM Tris-HCl, pH 7.6
- 150 mM NaCl
- 1% NP-40 (non-ionic deterget)
- 1% sodium deoxycholate (ionic deterget)
- 0.1% SDS (ionic deterget)
- 50 mM Tris, pH 7.4
- 250 mM NaCl
- 5 mM EDTA
- 50 mM NaF
- 1 mM Na3VO4
- 1% NP-40 (non-ionic deterget)
- 0.02% NaN3
|Compatible protein assays||BCA assays ||BCA assays |
|Recommended applications ||SDS-PAGE, Western blot ||ELISA, protein electrophoresis, Western blot|
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
, 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.
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.
Table 3. CytoWatch™ Series
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.
Table 4. Ordering information for laboratory consumbales and general laboratory equipment.
|CytoCite™ BG100 Portable Fluorometer||1 Fluorometer||CBG100|
|CytoCite™ Sample Tube *500 µL for Qubit® and CycoCite™ fluorometers*||500 Tubes||CCT100|
|ReadiUse™ 10KD Spin Filter||5 Filters||60502|
|ReadiUse™ Bio-Gel P-6 spin column||5 Columns||60500|
|PD-10 Spin Adapter||5 Adapters||60506|
|PD-10 Spin Adapter||10 Adapters||60507|
|PD-10 Buffer Reservoir||5 Pieces||60508|
|PD-10 Buffer Reservoir||10 Pieces||60509|
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.
Table 5. Properties and main applications of common detergents.
|SDS (sodium dodecyl sulfate)||Denaturing, strong lysis agent||Anionic||62||18,000||6-8 (0.17-0.23)||>100||No||Cell lysis, Electrophoresis, WB, Hybridization|
|Triton X-100||Non-denaturing, mild lysis agent||Non-ionic||140||90,000||0.24 (0.0155)||64||No||Cell culture, Enzyme immunoassays, IP, Membrane protein solubilization|
|Triton X-114||Non-denaturing, mild lysis agent||Non-ionic||-||-||0.21 (0.0113)||23||No||Cell lysis, Various routine protein and molecular biology techniques|
|NP-40||Non-denaturing, mild lysis agent||Non-ionic||149||90,000||0.29 (0.0179)||80||No||Isoelectric focusing|
|n-dodecyl-Β-D-maltoside||-||Non-ionic||98||50,000||0.17 (0.009)||-||No||Protein Crystallization|
|Brij 35||-||Non-ionic||40||49,000||0.09 (0.011)||>100||No||Cell lysis, Chromatography, Electrophoresis, HPLC, Membrane protein solubilization|
|Digitonin||Non-denaturing, mild lysis agent||Non-ionic||60||70,000||-||-||No||Membrane solubilization, Protein solubilization, Enzymology, Chromatography, Cell culture|
|Tween-20||Mild lysis agent||Non-ionic||-||-||0.06 (0.0074)||95||No||Cell lysis (mammalian cells), WB, ELISA, Enzyme immunoassays, Membrane protein solubilization|
|Tween-80||Mild lysis agent||Non-ionic||60||76,000||0.01 (0.0016)||-||No||ELISA, WB, Immunoassays|
|Octyl-beta-Glucoside||Non-denaturing, mild lysis agent||Non-ionic||27||8,000||23-24 (∼0.70)||>100||Yes||Membrane protein solubilization|
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
, can be slowed down significantly if samples are kept on ice or at 4°C and appropriate inhibitors are added to the lysis buffer.
Table 6. Properties of common protease and phosphatase inhibitors.
|Aprotinin||6511.5||Trypsin, chymotrypsin, plasmin||Reversible||10 mg/mL (H2O)||2 µg/mL||Dilute in water, 10 mg/mL. Do not re-use thawed aliquots.|
|Leupeptin||475.6||Lysosomal||Reversible||1 mg/mL (H2O)||5-10 µg/mL||Dilute in water. Do not re-use thawed aliquots.|
|Pepstatin A||685.9||Aspartic proteases||Reversible||1 mg/mL (MeOH)||1 µg/mL||Dilute in methanol, 1 mM|
|PMSF||174.2||Serine, cysteine proteases||Reversible||18 mg/mL (MeOH)||1 mM||Dilute in ethanol. You can re-use the same aliquot.|
|EDTA||372.2||Metalloproteases that require Mg2+ and Mn2+||Reversible||10 g/100 mL (H2O)||5 mM||Dilute in dH20, 0.5 M. Adjust pH to 8.0.|
|EGTA||-||Metalloproteases that require Ca2+||-||-||1 mM||Dilute in dH20, 0.5 M. Adjust pH to 8.0|
|Sodium fluoride||42.0||Serine/threonine phosphatases||Irreversible||40 mg/mL (H2O)||5-10 mM||Dilute in water. Do not re-use once defrosted.|
|Sodium orthovanadate||183.9||Tyrosine phosphatases||Irreversible||20 mg/mL (H2O)||1 mM||Dilute in water. Do not re-use once defrosted.|
Table 7. Ordering Information For Buffers, Solutions, and Lab Consumables
|5400||ReadiLink™ Protein Conjugation Stop Buffer||1 ml|
|5540||ReadiUse™ TCEP removal solution||1 ml|
|10000||10X Citrate Buffer *pH 6.0*||100 mL|
|11004||ReadiUse™ hydrogen peroxide solution *50 mM calibrated and stabilized solution*||5x10 mL|
|11010||Signal Guard™ HRP conjugate stabilizer||50 mL|
|11020||Signal Guard™ HRP reaction stopping solution||0.5 mL|
|17202||ReadiUse™ PCR Reaction Buffer||50 mL|
|20001||FluoroQuest™ Anti-fading Kit I *Optimized for Slide Imaging*||1 Kit|
|20003||FluoroQuest™ Anti-fading Kit II *Optimized for Plate Imaging*||1 Kit|
|20006||FluoroQuest™ Fluorescence Signal Enhancing Solution||1 Kit|