Instrumentation & Methodology of Electrophoresis

Instrumentation of Electrophoresis

Power Supply

Electrophoresis is carried out under constant voltage or current or power. The migration rate should be kept constant through a constant power supply. If the increased current is supplied, there is increased heat production through resistance, which causes thermal agitation of dissolved ions. There will be increased evaporation of water from the apparatus. This will lead to an increase in the concentration of ions in the buffer.

Buffers

Buffer establishes the electrical charge on the solute and pH of the system. It is used in carrying the electrical current.

The ideal buffer:

• Should not interfere in the detection of desired analytes
• Maintaining the solubility of analytes
• Maintaining the buffering capacity throughout the analysis process
• Producing the desired range of separation

Citrate, acetate, formate, and phosphate are acidic buffers used for lower pH. Tris, tricine, and borate are basic buffers used for maintaining high pH. The buffers which are used are made up of monovalent ions as the valency (ionic strength) and molality is equal. The prepared buffers can serve as a good platform for the growth of microorganisms, so they should be properly refrigerated when they are not used. Cold buffer can be used in the process as it provides increased resolution of samples and decreased evaporation of the solvent in the process. Large volumes of the buffer can be reused up to four times, but the smaller volumes can be discarded immediately. The higher ionic strength of the buffer is useful in producing a sharper resolution of samples, though there is a risk of damage to heat-labile compounds as heat is generated much.

Support Media

Slabs, sheets, columns of starch, polyacrylamide, agarose, the membrane of cellulose acetate are used as supporting media. It is in a colloid form, made up of more than 90% of water.  It acts as a molecular sieve, through which the separation of molecules is done. It is porous, allowing small molecules to pass through while obstructing the movement of larger molecules. It should be electrically neutral. Nowadays, agarose gel is mainly used as a support medium to carry out electrophoresis.

Starch Gel

Separates biomolecules on the basis of molecular size and surface charge

It is not used now as preparation of gel showing reproducibility is pretty difficult

Cellulose Acetate

• It is prepared by treating acetic anhydride with cellulose.
• It is required to be soaked in the buffer before use, so as to get the softened form
• Before taking densitometry, it requires cleaning
• It is not very much useful in clinical application.

Agarose

The separation of molecules is based only on mass to charge ratio as all proteins can pass through the pore of the gel. It is capable of separating proteins into five zones only. There is a lower affinity for proteins, which provides good clarity after drying. Endosmosis is negligible and it is devoid of ionizable groups, being neutral. The downside is the recovery of DNA is affected by inhibitors.

Polyacrylamide

• It is not charged and no endosmosis takes place.
• It is able to accommodate a large volume of protein in a single slot.
• DNA recovery is not affected by inhibitors.
• Separation is based on both molecular weight and mass to charge ratio.
• There is no diffusion of larger proteins like lipoproteins, fibrogen through the pores as the pore size is relatively higher.
• Able to separate proteins into twenty different zones
• Useful in the study of individual proteins like isoenzymes.
• It is transparent, durable, thermostable and chemically inert comparatively.
• Being carcinogenic is a con.

Electrophoresis Chamber

It is a plastic box or tank, which is filled with a buffer for the migration of biomolecules. It has a transparent cover, which facilitates easy viewing of the migration process. It is connected to a power source.

Containers for staining and destaining gel

The trays and containers are used for the purposes of staining and destaining gels. Both open form and lidded boxes are available. Generally, they have a polypropylene base. They are tightly fitted and clear. They are resistant to stains and solvents.

Electrodes

There are two platinum electrodes, which facilitate the separation of molecules by attracting opposite charges. An anode is used for binding to positive ions and a cathode binds negative ions.  

Gel Caster and Comb

The gel is poured after dissolving in the solvent into a gel caster, which holds the gel and is kept inside the apparatus. A comb is used to put the wells for the loading of samples.

Methodology of Electrophoresis

• Gel is prepared by dissolving in boiling water.
• Solution is cooled to a warmer temperature and poured into a mold or caster.
• After the gel is solidified, a comb is used to put wells in it.
• Then the gel is placed in the electrophoretic chamber.
• Buffer is filled in the chamber not exceeding 1/3rd of the total volume of the chamber.
• Sample is mixed with loading dye, either with fluorescent tag or ethidium bromide as dye adds density to the sample and provides color to it.
• Sample is loaded into the wells using a clean micropipette.
• Molecules migrate towards oppositely charged electrodes, thus DNA, being negatively charged, moves towards anode. The migration is visually tracked via dye.
• After the process ends, gel is stained and visualized using a gel imager instrument.
• Sizes of sample fragments are calculated through logarithm of molecular weight, by comparing with the standard. DNA concentration is measured by taking absorbance at 260 nm, which is compared with fluorescent standard having a particular intensity and intensity is directly proportional to concentration.

Conclusion

Each and every part of the electrophoretic apparatus is useful and significant in one or another way. Depending on the type of electrophoresis, the reagents used and parts may vary. Concentration may be varied depending on the status of the sample. The methodology for the technique is given, yet it differs slightly for different types of electrophoresis. While doing the experiment, following optimized protocol is recommended.