Pros, Cons & Applications of Electrophoresis

Zone Electrophoresis

Pros

• Small quantity of samples is only needed.
• Cheap and easy maintenance.

Cons

• Not suitable for isoelectric point determination and accurate mobility.

Applications

• Useful in biochemical investigation.

Thin layer electrophoresis

Pros

• Good resolution
• Less time needed for the process

Applications

• Two dimensional study of nucleic acid and protein hydrolysate.
• Combined with electrophoretic chromatography.

Cellulose acetate electrophoresis

Pros

• No tailing of hydrophilic materials and proteins.
• Available as wide variety of thickness layers and particle sizes
• Good resolution and sharp bands
• High voltage enhances the resolution

Cons

• Expensive
• Electroosmosis is induced by carboxylic and sulfonic residues.

Applications

• Serves as an alternative to paper electrophoresis.
• Analysis of biological and clinical protein samples like globulins and albumins

Paper electrophoresis

Pros

• Economical
• Easy to use

Cons

• Electroosmosis happens
• Not able to resolve proteins and hydrophilic molecules because of the ionogenic and adsorptive nature of paper, resulting in distortion and tailing of component bands.

Applications

• Serum analysis
• Clinical disease like sickle disease, hemoglobin abnormalities
• Separation of blood clotting factor
• Detection of adulteration in drugs

Gel electrophoresis

Agarose Gel

Pros

• Preparation is easy
• Small concentration of agar is required
• Huge quantities of samples can be separated and recovered.
• Due to less adsorption, sharp zones are obtained
• Resolution is better than that of filter paper chromatography
• Good method of preparative purpose as there is good recovery of protein
• Negatively charged protein molecules’ adsorption is less.
• Adsorption of proteins is lesser compared to other methods

Cons

• High electro osmosis
• Compared to Polyacrylamide gel electrophoresis, resolution is lesser
• Results are different due to different batches and sources of agar and hence, purification is essential.

Applications

• Separation of different types of nucleic acids and proteins
• Used in immunoelectrophoresis

Starch

Pros

• Sharp zones and high resolving power
• Used as preparative and analytical electrophoresis
• Resolved proteins can be recovered and reasonable yield is obtained.

Cons

• Electro Osmosis occur
• From batch to batch, variation of pore size takes place
• Degassing needed

Applications

• Protein separation

Polyacrylamide Gel

Pros

• Over broad range of pH and temperature, the gel is stable
• Different pore sizes in gels can be prepared
• Relatively, the separation speed is better

Cons

• Waiting time is longer for the gel to set
• Gas bubbles should be avoided while pouring the gel
• Presence of free acrylamide in polyacrylamide gel, which is a potent neurotoxin

Applications

• Estimation of molecular weight of nucleic acids and proteins
• Discovery of proteins’ subunit structure
• Isolated proteins’ purification
• In body fluids, monitoring the change in protein content
• Identification of disulfide bonds between proteins
• Quantification of proteins
• Blotting
• Checking purity of proteins

Moving Boundary Electrophoresis

Pros

• Without the use of denaturing agents, recovery of biologically active molecules can be performed.
• For measurement of electrophoretic mobility, it serves as a  reference.

Cons

• Expensive
• Optical system with elaborate set up is required.

Applications

• Macromolecular system’s homogeneity can be studied.
• Used for analyzing complex biological molecules.

Isotachophoresis

Pros

• No sample preparation needed
• Speedy result
• Applicable to broad spectrum of samples

Applications

• Separation of inorganic substances and proteins
• Analysis of inorganic ions in amino acids, proteins and water
• Analysis of anions in serum and union
• Analysis of organic acids in silage

Isoelectric focussing 

Pros

• Because of applied field and pH gradient, band spreading is minimized and high resolution can be attained.
• Even 0.001 pH difference in proteins is detected and proteins are separated.

Cons

• Higher voltage of 2000 V is required as carrier ampholytes are used in higher concentration. Hence, the  electrophoretic apparatus must be cooled, which is difficult sometimes.

Applications

• Separation of peptides and proteins
• Utilized for research in taxonomy, enzymology, cytology and immunology.

Capillary Electrophoresis

Pros

• High efficiency in separation
• Simple and automated
• Shorter analysis time
• Lower sample volume is enough
• Easy to operate
• Able to separate both uncharged and charged molecules
• Various mechanisms for selectivity
• Lower cost
• Use of aqueous solvent rather than organic solvent, hence environment friendly

Cons

• Sensitivity and resolution limits

Applications

• Determines ions in saliva
• Detection and amplification of DNA fragments
• Detection of microbial and bacterial contamination

Immunoelectrophoresis

Pros

• Due to electric field and pH gradient, higher resolution is obtained

Cons

• Higher voltage of 2000 V is required as carrier ampholytes are used in higher concentration. Hence, the  electrophoretic apparatus must be cooled, which is difficult sometimes.

Applications

• Used in immunology for protein identification.
• Identification and quantification of proteins in the serum
• Used in the patients suspected with polyclonal and monoclonal gammopathies.
• Detection of abnormal and normal proteins like myeloma in human serum
• Used in the diagnosis of multiple myeloma and hypogammaglobulinemia
• Monitoring purity of antigen-antibody and antigen
• Identification of single antigen in antigen’s mixture

Applications of Electrophoresis

• Agricultural testing
• Medical research
• DNA sequencing
• Protein purification and research
• Food industry
• Analysis of antibiotics, terpenoids and steroids
• Testing the purity of thyroid hormones 
• Separation of free insulin from plasma proteins
• Diagnosis of diseases like liver and kidney
• Separation of vitamins and carbohydrates
• Quantitative separation of enzymes, antibiotics and RBC
• Fractions of cells can be quantitatively separated
• Separation of ephedrines and scopolamine
• Studying binding of iron to serum proteins when combined with autoradiography
• Separation of insulin, nucleic acids, phenols, amino acids, alcohols, alkaloids, carbohydrates and organic acids
• Utilized in biochemical and clinical fields for the study of protein mixture like hemoglobin, blood serum
• Study of antigen-antibody interactions.

Conclusion

We have come to know about the advantages and disadvantages exhibited by each and every type of electrophoresis. Applications of each type of electrophoresis have also been discussed. Some types of electrophoresis are suitable for some applications and a few are compatible with a particular application. Cost involved and time taken for the completion of the process also play a critical role in the selection of a particular type of electrophoresis.