Electrophoretic techniques for Bio-medical Researchers

Meaning of electrophoresis

Electrophoresis is a separation technique that is based on the movement of charged particles in an electric field. The term ‘electrophoresis’ was coined from the Greek word ‘phoresis’, which means ‘being carried’. Electrophorosis literally means ‘to carry with electricity’

Discovery of electrophorosis

In 1807,Russian physicist Alexander Reuss identified the migration of colloidal particles in an electrical field. In 1879, Hermann von Helmholtz generalized the experimental observations into an equation of electrophoretic principles. In 1930, Swedish chemist Arne Tiselius described the first electrophoretic system. He received the Nobel prize for chemistry in 1948 for his work.

Definition of electrophoresis 

Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field.

Principles of electrophoresis

Electro-migration 

At any given pH , the electrically charged molecules may exist in solution either as cations (+) or anions (-).Negatively charged molecules move to the anode(+).Positively charged molecules move to the cathode(-).Highly charged molecules move faster towards the electrode of opposite charge than those with lesser.
Electro-osmosis

The movement of entire fluid near wall of capillary in one direction!
anode (+ve) -> cathode (-ve)
Electromotive force(EMF) 

Electrophoresis is based on electromotive force(EMF) that is used to push or pull the molecules through the gel matrix. By placing the mixture of molecules in wells in the gel and applying an electric current, the molecules will migrate through the matrix. The separated molecules take directions based on the total electric charges.
Principle of velocity of migration of separated molecules

Velocity of migration of the molecules, v=E.q / f
Where E=electric field in volts/cm; Q=the total electric charge on the molecule; F=the frictional coefficient which is a friction of the mass and shape of the molecule.

Electrophoretic Theory

Two laws are relevant to the use of power supplies for electrophoresis of macromolecules: Ohm’s law and second law of electrophoresis.

 Ohm’s Law - Current (I)=Voltage (V)/Resistance (R)

Ohm’s Law states that current is directly proportional to the voltage and is inversely proportional to the resistance. Resistance of the system is determined by the buffers used, the type and configurations of the gels being run, and the total volume of all the gels being run.

      Second Law of electrophoresis - Watts (W)=Current (I) x Voltage (V)

     The Second Law states that power or watts (a measure of the heat produced) is equal to the               product of the current and voltage.  Since V=I x R, this can also be written as Watts=I² x R.

Factors influencing rate of migration of ion

The migration of ions in an electric field depends on the net charge of the molecule, size and shape of the molecule, buffer pH, strength of electrical field, properties of support media and Temperature of the operating system.

Buffers for electrophoresis

Barbitone buffer – (around 8.0 pH)- serum protein separation , poor resolution, weak buffer.
Phosphate buffer ( around 7.0 pH) - Enzyme separation,low buffering capacity.- high conductivity
Tris – borate – EDTA buffer (TBE) -(pH around 8.0) - Nucleic acid Separation,Good resolution , high buffering capacity , low conductivity.
Tris – acetate – EDTA buffer (TAE)- (pH around 8.0) - Nucleic acid separation, high resolution , high buffering capacity , low conductivity.
Tris – glycine buffer -(pH more than 8.0)- Protein separation, high buffering capacity , low conductivity

Support media for electrophoretic run

Paper – poor conductor of electricity absorbate proteins, non - transparent poor resolution.
Agar- flow of solvent electro endosmosis, vary thickness , transparent poor resolution
Cellulose acetate strip- tailing of bands poor resolution non-absorbing.
Starch- form opaque gels non-absorbing high resolution
Agarose -highly transparent porous – high resolution east preparation
Acrylamide – stable , non –reactive highly transparent.

Kinds of electrophoretic techniques

Zonal electrophoresis - Consden,Gordon and Martin in 1946 introduced this technique. Sample is applied as a narrow band. Separation occurs discrete bands. Numerous support media –paper, cellulose acetate, agar gel starch gel and acrylamide gel can be used.
Paper electrophoresis - This technique was introduced by Durrum (1950), Flynn and Mayo (1951). A small volume of the sample is placed evenly along a line drawn across a strip of Whatmann paper previously soaked in buffer. The ends of the paper are soaked in buffer solutions. Passage of electricity cause separation.
Starch gel electrophoresis - Starch matrix is suitable for isoenzymes . Partially hydrolysed potato starch is used. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining.
Cellulose acetate electrophoresis - Kohn (1957-1961) introduced this technique. Strips of cellulose acetate are used.Better resolving power. No absorption of proteins. No trailing. Excellent separation of plasma proteins, transparent.
Gel electrophoresis - Electrophoresis through agarose or polyacrylamide gels is a standard method used to separate, identify and purify nucleic acids. Gel electrophoresis involves the use of a gelatinous material such as agarose, acrylamide, starch or cellulose acetate as the matrix. The gel acts as a support medium for the sample. Gels are used to separate samples containing proteins or DNA.

     Starch Gel -- swollen potato starch granules.

     Agarose Gel is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool. 

      Polyacrylamide Gels -Polyacrylamide gel is made chemically by acrylamide (the monomer) and bisacrylamide (the cross-linker) catalyzed by initiator (amonnium persulfate or  riboflavin)  and accelerator (TEMED). Acrylamide can be polymerized into any desired shape :

•        Tube Gels -- polymerize in glass tubing ==> cylindrical shape

•        Slab Gels -- polymerize between glass plate 

Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes.

•        The separation medium is a gel made from agarose, which is a polysaccharide derivative of agar.

•        The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon charge, size and shape.

•         These characteristics,together with buffer conditions, gel concentrations and voltage, affect the mobility of molecules in gels.

  Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis 
 SDS- PAGE is a most widely used technique for analysis and characterization of proteins and nucleic acids.

•        Sample preparation – The protein sample is heated at 100⁰C in a dilute solution sodium dodecyl sulfate .This breaks down all native quaternary, tertiary, and secondary structures. Then b-mercapto ethanol is added to cleave the disulfide bonds.

•        Gel preparation – the polymerization is initiated by ammonium per sulfate or riboflavin. N-tetramethyl ethylene diamine (TEMED) catalyses the formation of free radicals from     persulfate which in turn initiate polymerization. Gels ranging from 3 to 30% acrylamide concentration can be made and can be used for the separation of molecules up to 1x10⁶ datons.

•        Sample application –about 2 µg of the sample is loaded in each well. Over loading of samples decrease the resolution of bands.

•        Marker dyes – to follow the sample tracking a marker dye e.g. bromophenol blue gives color. After run the gel was stained with the dye coomasie blue and photographed.

Applications of PAGE –

1.    PAGE is used to estimate molecular weight of proteins and nucleic acids.

2.    PAGE is used to determine the subunit structure of proteins.

3.    PAGE is used to purify isolated proteins.

4.    PAGE is used to investigate various liver and kidney diseases by analyzing human serum proteins.

5.    PAGE is used to monitor the changes in protein content in body fluids.

q Continuous - discontinuous  gel systems –

q  Continuous  system--gel and tank  buffers are the same, single phase  gel; examples are PAGE, agarose,  and starch gels.

q Discontinuous system--gel and  tank buffers are different, two phase gel (stacking gel); example  is PAGE.

Discontinuous polyacrylamide gel electrophoresis –DISC-PAGE -

Two gel systems – a stacking gel and a running gel

Several buffer systems

·         Cathode -  Tris – glycine 8.6 pH

·         Wells – Tris – Cl 6.5 pH

·         Stacking gel – Tris – Cl 6.5 pH

·         Separating gel – Tris – Cl 8.7 pH

·         Anode  - tris – glycine 8.

Generation of voltage discontinuity

Uses of electrophoresis techniques 

•         Human DNA can be analyzed to provide evidence in criminal cases, to diagnose genetic diseases, and to solve paternity cases.

•         Samples can be obtained from any DNA-containing tissue or body fluid, including cheek cells, blood, skin, hair, and semen.

•        A person’s “DNA fingerprint” or “DNA profile” is constructed by using gel electrophoresis to separate the DNA fragments from several of its  highly variable regions.

•        Conservation biologists use DNA profiling to determine genetic similarity and kinship among populations or individuals.