BUFFERS

 BUFFERS

A buffer is a substance that can keep constant concentrations of the hydrogen ions in a solution where a small amount of acid or base is introduced. Buffers are essential since they provide a strictly controlled pH environment and thus give a conducive environment for the investigation of biomolecules. A lab provides the controlled conditions required to research biomolecules.

Phosphate buffers are widely used since they provide a condition that is closer to physiological conditions. The phosphate buffers have a pH of 6.5 to 7.5. Phosphate buffers form precipitates with ions such as Ca²⁺ and Mg^2+, which are essential in the functioning of many biomolecules (Wilson, D.J. (2016)).

Zwitterionic buffers are chemicals that contain both positive and negative charges in their species. Zwitterionic buffers have gained an extensive use in biological samples because they are very soluble in aqueous solutions and have a reduced ion effect that allows preparation of diluted buffers from stock solutions (Brown, K.A., Sharifi, S., Hussain, R., Donaldson, L., Bayfield, M.A. and Wilson, D.J. (2016)). Buffers such as Tris, HEPES, and PIPES can produce radicals under a variety of experimental conditions and thus should be avoided in the biological redox process or radical-based reactions.

Carboxylic acid buffers provide a buffering capacity in the pH range of 3 to 6. Their use is limited due to interference with the process under investigation since they are naturally found in all biological systems. Citrate and succinate can form metal complexes that can affect the activity of enzymes and proteins that use these metals for an activity or structural ability.

Table 1. Table of buffers and their pH value.

The sodium phosphate buffer has many uses since it is isotonic and non-toxic to most cells. It is commonly used in biological research. Additionally, the sodium phosphate buffer is used in substance dilution, and the drying of biomolecules since water molecules within it will be structured around the substance for immobilization to a solid substance. Sodium phosphate buffers are capable in cell container rinsing. The sodium phosphate buffer, when mixed with EDTA, ca be used to disengage attached and clumped cells.

The resolving buffer is used to separate and analyze macromolecules based on their size and charge. In clinical chemistry, resolving buffer is used to separate proteins by charge or size. In biochemistry and molecular biology, it is used to separate the mixed population of DNA and RNA fragments based on length, it estimates the size of DNA and RNA fragments or to separate proteins by charge. When proteins are fixed in polyacrylamide gels, the resolving buffer prevents diffusion of the proteins; hence it keeps the bands sharp and correctly resolved. The resolving gel is used in the conservation of agarose when fewer samples need to be run utilizing a wall comb.

The stacking buffer can be used as an embedding medium for sectioning tissues. The ability of the stacking buffer to concentrate proteins in a sample enables it to be used to improve the resolution of the electrophoresis. The stacking buffer also helps clean the sample from the Cl^– ions from the Tris-HCI buffer. It can do so because the stacking buffer has a higher electrical resistance and a higher electric field that provokes the faster movement of every charged particle in the gel and proteins.

The running buffer with SDS is used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins. It is used as the anode and the cathode (Rodger, K., 2001). The running buffer’s electrical charge enables the separation of proteins, DNA, and RNA for analysis, and it does so while maintaining pH levels. The running buffer is used for the separation of charged molecules by their molecular masses in an electric field. When used with RunBlue Precast Gels, the running buffer can provide the best running conditions and optimal sample resolution. The buffer system gives improved resolution at low molecular weights while retaining an excellent resolution at high molecular weights, and it can be used for reduced and non-reduced samples.

The running buffer without SDS is used as a reducing agent that should be added during sample preparation. With the use of the running buffer without SDS, one can separate proteins based on their size, net charge, and shape. Development of purified, active proteins is done by use of the running buffer without SDS. The buffer is used because no denaturants are used; thus, subunit interactions within a multimeric protein are retained, and one can gain quaternary structure information. High-resolution native electrophoresis is done with the help of the running buffer without SDS.

REFERENCES

Rodger, K. et al. 2001. Biotechnol. Appl. Biochem. 33:209-214. PMID:11389675.

Brown, K.A., Sharifi, S., Hussain, R., Donaldson, L., Bayfield, M.A., and Wilson, D.J. (2016) Distinct Dynamic Modes Enable the Engagement of Dissimilar Ligands in a Promiscuous Atypical RNA Recognition Motif. Biochemistry 50, 7141–7150.

Wilson, D.J. (2016) Millisecond Hydrogen Exchange, in Hydrogen Exchange Mass Spectrometry of Proteins (Weis, D.D., Ed.) 1st ed., pp 73-88, Wiley, New Jersey.