Electrophoresis: Introduction, Factors Affecting, Basic Components, Buffers, Supporting Media and Common Dyes
Introduction of electrophoresis
Electrophoresis is the migration of a charged solute or particle of any size in a liquid medium under the influence of an electric field.
Biological molecules like amino acids, peptides, proteins, nucleotides, and nucleic acids exist in a solution as electrically charged particles at a given pH. When electricity is applied to such a medium, depending on their net charge and molecular size, the particles will migrate deferentially.
Factors affecting analyte migration
The net charge of the molecule
Size and shape of the analyte
Solvent viscosity
the pH of the solution
Properties of the supporting medium
Electroendosmosis
Temperature
Wick flow
Electrophoretic mobility (μ) = rate of migration(cm/s) per unit field strength(volts/cm)
The basic component of the Electrophoretic system
Electroscopes unit
Power supply/pack
Buffers
Supporting media
Tracking dye
Staining dyes/another tracing system
Visualization
Electrophoresis system
Vertical gel system
Horizontal gel system
Buffers of electrophoresis
Depending on the nature of the sample and the experimental objective essential to maintain a constant state of ionization of the molecules being separated.
Commonly employed buffers:
Phosphate buffer: around 7.0 pH
TBE (Tris Borate EDTA) buffer: around 8.0 pH
TAE (Tris Acetate EDTA) buffer: above 8.0 pH
TG (Tris-Glycine) buffer: more than 8.5 pH
TCE (Tris Citrate EDTA) buffer: around 7.0 pH
TE (Tris EDTA) buffer: around 8.0 pH
TME (Tris Maleic acid EDTA) buffer: around 7.5 pH
Lithium Borate (LB) buffer: around 8.6 pH
Support media for electrophoresis
It provides the matrix in which molecules separation takes place
inert chemically, easily available, high electrical conductivity, low absorptivity, desirable sieving effect, controlled porosity, high transparency, low electroendosmosis, moderate to high rigidity, feasible preservation, low toxicity, and easy preparation. e.g.
Paper: Filter paper, cellulose acetate strips
Gels: starch, agarose, polyacrylamide
Dyes of electrophoresis
Tracking dyes (Bromophenol blue, xylene cyanol, cresol purple, Orange G) along with sucrose, glycerol, or Ficoll which make the solution dense.
Staining dyes: Coomassie Brilliant Blue, silver stain, Amido black, Kenacid blue for proteins Ethidium Bromide (EtBr), SyBR green, propidium iodide for DNA
Visualization: a UV light source
Further Readings
- Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th Edition
- Principles and Techniques of Biochemistry and Molecular Biology, Wilson and Walker 7th Edition
- Short protocols in molecular biology, 4th edition
- Pulsed-field gel electrophoresis( A practical Guide )by Bruce Birren21.
- Molecular Biology. Editor: David Freifelder, 2nd ed, Publisher Jones and Bartlett Inc.