Electrophoresis – A brief review

Electrophoresis is the movement of charged particles through an electrolyte when subjected to an electric Field-

• Cations move towards the cathode
• Anions move towards the anode 
• By this technique, solutes are separated by their different rates of travel through an electric field.
• Commonly used in biological analysis, particularly in the separations of proteins, peptides, and nucleic acids 

Factors affecting Electrophoresis

The rate of migration of a solute in an electric field depends on the following factors-

1. The net charge on the particle
2. Mass and shape of the particles 
3. pH of the medium
4. Strength of electric field
5. Properties of supporting medium
6. Temperature

Electrophoretic Mobility 

 Electrophoretic mobility is defined as the rate of migration (cm/sec)per unit field strength(volts/cm)

 µ=Q/6πrη

Where µ- Electrophoretic mobility

Q-Net charge on the ion

the r-Ionic radius of the solute

η- Viscosity of the medium

• The Electrophoretic  mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium
• The pH of solution affects the mobility of the ion by determining the amount and nature of the charge
• Proteins, nucleic acids, nucleotides and amino acids  bear charged polar groups making them suitable groups for electrophoresis
• Carbohydrates carrying no charged groups are first bound to charged groups like Borate or Sulfite ions and then electrophoresis is carried out. 
• Lipids are not electrophoresed because electrophoretic current requires polar solvents in which most lipids are insoluble

Types of Electrophoresis

1) Horizontal

2) Vertical

  Vertical electrophoresis is mainly used for Polyacrylamide gel electrophoresis.

Electrophoresis Apparatus

  Electrophoresis apparatus consists of-

1. Buffer tank -to hold the buffer
2. Buffer
3. Electrodes- made of platinum or carbon
4. Power supply
5. Support media

Figure-1- Showing apparatus for Horizontal electrophoresis.

Note-Choice of the buffer depends on the nature of substance to be separated and the electricity is supplied at a constant current and voltage. 

The electrophoresis support on which separation takes place may contact the buffer directly or by means of wicks.

  The entire apparatus is covered to minimize separation

Support media for electrophoresis

1. Filter Paper
2. Cellulose acetate membrane
3. Agar or Agarose gel
4. Starch Gel
5. Polyacrylamide gel

A)  Paper Electrophoresis

 The support medium is a filter paper frequently used in isolating proteins, amino acids and oligopeptides.

Procedure–

1. A long strip of filter paper is moistened with a suitable buffer      solution of the desired p Hand the sample is applied transversely across the central part of the strip
2. Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes
3. The electric field of about 20 volts/cm is established
4. The charged particles of sample migrate along the strip towards respective electrodes of opposite polarity, according to net charges, sizes, and interactions with the solid matrix
5. A homogeneous group of particles migrates as a separate band
6. The electrophoresis is carried out for16-18 hours
7. Separated Proteins are fixed to a solid support using a fixative such as Acetone or Methanol
8. Proteins are stained to make them visible
9. The separated proteins appear as distinct bands
10. Drawback-long time interval and blurring of margins

B) Cellulose Acetate Membrane Electrophoresis

• Preferred solid support medium
• Less time-consuming 
• Excellent separation 
• Membranes can be stored for a longer time 
• Widely used for separation of lipoproteins, isoenzymes, and hemoglobin

C) Gel Electrophoresis

• The term “gel” in this instance refers to the matrix used for containing and then separating the target molecules
• In most cases, the gel is a crosslinked polymer whose composition and porosity are chosen based on the specific weight and composition of the target to be analyzed. 
• A  gel block made of Polyacrylamide, Agarose or substituted starch gel is used in this method as the solid support
• Agar gel is used for separation of different types of protein mixtures as well as nucleic acids 
• Polyacrylamide is most suitable for the separation of nucleic acids. It is also frequently used in separating proteins, peptides and amino acids from microgram quantities of mixed samples

a) Agarose gel electrophoresis

• Commonly used support medium
• Less expensive than cellulose acetate 
• Equally good separation 
• Agar is a complex acidic polysaccharide containing monomers of sulfated galactose
• Agarose is  a sulfate-free fraction of Agar 
• The gel is prepared in the buffer and spread over a microscopic slide 
•  A small sample of serum or biological fluid is applied by cutting into the gel with a sharp edge 
• The electrophoretic run takes about 90 minutes

b) Poly Acrylamide Gel Electrophoresis-PAGE

• Most popular type
• Polyacrylamide is a polymer formed when acrylamide is heated with a variety of catalysts with or without cross-linking agents 
• It is thermostable, transparent, strong and relatively chemically inert
•  Gels are uncharged and are prepared in a variety of pore sizes
• Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving

Types of PAGE

PAGE can be classified according to the separation conditions into:

  Native-PAGE:

•  Separation is based upon charge, size, and shape of macromolecules
•  Useful for separation and/or purification of the mixture of proteins 
• This was the original mode of electrophoresis.

Denatured-PAGE or SDS-PAGE

•  Separation is based upon the molecular weight of proteins
• The most common method for determining MW of proteins 
• Very useful for checking the purity of protein samples

PAGE-Procedure

1. The gel of different pore sizes is cast into a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom(figure-2)
2. Micrograms quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such a pH so as to change sample components into anions
3. The foot of the gel column is made to dip in the same buffer in the bottom reservoir
4. Cathode and anode are kept above and below the column  to impose an electric field through the column
5. Macromolecular anions move towards the anode  down the gel column
6. There is no external solvent space, all the migratory particles have to pass through the gel pores 
7. Rate of migration depends on the charge to mass ratio
8. Different sample components get separated into discrete migratory bands along the gel column on the basis of electrophoretic mobility and gel filtration effect
9. PAGE may yield 20 or more fractions and may be used to study individual proteins and nucleic acids in serum especially genetic variants and isoenzymes

 Figure-2- Polyacrylamide gel electrophoresis

Slab PAGE

• The Polyacrylamide gel is cast as a thin rectangular slab inside a plastic frame and this slab is placed vertically on a buffer solution taken in a reservoir 
• Several samples dissolved in dense sucrose solution or glycerol are placed in separate wells cut into the upper edge of the slab and are covered by the same buffer solution. Cathode and anode are above and below to produce electric field effects. Different components migrate simultaneously down parallel lanes in the slab and get separated into bands

SDS PAGE

• SDS-PAGE, sodium dodecyl sulfate Polyacrylamide gel electrophoresis, is technique widely used in biochemistry, forensics, genetics , and molecular biology to separate proteins according to their electrophoretic mobility 
• The SDS gel electrophoresis of samples having an identical charge to mass ratios results in fractionation by size and is probably the world’s most widely used biochemical method 
• When a detergent  SDS(Sodium-Dodecyl-Sulfate)is added to PAGE the combined procedure is termed as SDS PAGE SDS coats protein molecules giving all proteins a constant charge-mass ratio
• Due to masking of charges of proteins by the large negative charge on SDS binding with them, the proteins migrate along with the gel in order of increasing sizes or molecular weights 
•  The molecular weight of a given protein can be determined by comparing the relative electrophoretic mobility of sample with that of standard protein of known molecular weight when both the sample and the standard proteins are electrophoresed side by side in the same gel slab (figure-3)
• In oligomeric proteins, SDS PAGE usually gives the molecular weight of separated monomer chains of the proteins because SDS cleaves the non-covalent bonds interlinking the monomer chains in the intact molecule. 

 Figure-3- SDS PAGE

VISUALIZATION

• After the electrophoresis is complete, the molecules in the gel can be stained to make them visible
• Ethidium bromide, silver, or coomassie blue dye may be used for this process 
• Other methods may also be used to visualize the separation of the mixture’s components on the geL 
• If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

OTHER TYPES OF ELECTROPHORESIS

 Capillary Electrophoresis

 Immuno electrophoresis

 Isoelectric focusing (Isoelectrophoresis)