- Gel electrophoresis is a commonly used technique for resolving proteins or nucleic acids.
- In general, a sample of one particular type of macromolecule (protein, DNA, or RNA) is placed in a well at or near the end of a gel matrix (gel).
- The composition of an electrophoresis gel is a semisolid open meshwork of interlinked linear strands.
- A gel is cast as a thin slab with a number of sample wells.
- After the wells of a gel are loaded with sample, an electric field is applied across the gel, and charged macromolecules of the same size are driven together in the direction of the anode through the gel as discrete invisible bands of material.
- The distance that a band moves into a gel depends on the mass of its macromolecules and the size of the openings (pore size) of the gel.
- The smaller macromolecules travel further than the larger ones.
- The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel.
- The tracking dye is a charged, low-molecular-weight compound that is loaded into each sample well at the start of a run.
- When the tracking dye reaches the end of the gel, the run is terminated.
- The bands, which are aligned in a lane under each well, are visualized by staining the gel with a dye that is specific for protein, DNA, or RNA.
- Discrete bands are observed when there is enough material present in a band to bind the dye to make the band visible and when the individual macromolecules of a sample have distinctly different sizes. Otherwise, a band is not detected.
- If there is little or no difference among the sizes of the macromolecules in a concentrated sample, a smear of stained material is observed.
- The intensity of a stained band reflects the frequency of occurrence of a macromolecule in a sample.
- The molecular mass (molecular weight) of a gel-fractionated macromolecule (band) is determined from a standard curve that is based on a set of macromolecules of known molecular mass (size markers) that covers the separation range of the gel system and is run in one or both of the outside lanes (calibrator lanes) of the same gel as the samples.
- The logarithm of the molecular mass of a size marker is related to its relative mobility (Rf) through a gel.
- The value of (Retention factor)Rf is defined as the distance traveled by a band divided by the distance traveled by the tracking dye (ion front).
Rf = migration distance of the protein ÷ Migration distance of the dye front
- The relationship between the logarithm of the molecular mass of each size marker and its Rf value is
plotted. Then, with this standard curve, a molecular mass is calculated for each band in a lane.
- The units of molecular mass for proteins and double-stranded and single-stranded nucleic acids are daltons, base pairs, and bases, respectively.
- The size markers are included in the same gel as the samples because the extent of mobility of a macromolecule(s) varies from one electrophoretic run to the next.
- Polyacrylamide is the preferred gel system for separating proteins.
- Copolymerization of monomeric acrylamide and the cross-linker bisacrylamide forms a lattice of crosslinked, linear polyacrylamide strands.
- The pore size of a polyacrylamide gel is determined by the concentration of acrylamide and the ratio of acrylamide to bisacrylamide.
- For many applications, a protein sample is treated with the anionic detergent sodium dodecyl sulfate (SDS) before electrophoresis.
- The SDS binds to proteins and dissociates most multichain proteins.
- Each SDS-coated protein chain has a similar charge-to-mass ratio.
- Consequently, during electrophoresis, the separation of the SDS–protein chains is based primarily on
size, and the effect of conformation is eliminated.
- SDS–polyacrylamide gel electrophoresis with a 10% polyacrylamide gel resolves proteins that range from 20 to 200 kilodaltons (kDa).
- Agarose, which is a polysaccharide from seaweed, is used routinely as the gel matrix for the electrophoretic separation of medium-size nucleic acid molecules.
- A 1% agarose gel can resolve duplex DNA chains that range from about 600 to 10,000 bp.
- Specialized agarose gel electrophoresis systems are available for fractionating DNA molecules with millions of base pairs, denatured DNA, and denatured RNA.
- In addition, for specific purposes, polyacrylamide gels are used for separating DNA molecules.
- For example, DNA chains that are as small as 6 bases and that differ from each other by 1 nucleotide can be resolved with a 20% polyacrylamide gel.