GEL ELECTROPHORESIS

Introduction

Gel electrophoressis is a separation technique that uses an electric field to separate macromolecules based on the physical properties of molecule size/mass and electrical charge. Nucleic acids and proteins are the molecular mixtures typically sorted through gel electrophoresis.

The technique uses an applied electrical field to move molecules across a gel block. The physical attributes of the molecules in a sample dictate the speed with which they will migrate through the gel matrix.

Gellin' Like a Fellon

The type of gel used in gel electrophoresis depends on the types of organic molecules being separated. In studies looking at large molecules, such as nucleic acids of more than a few hundred bases in length, agarose gel is used because it has a large pore size. Agarose is a polysaccharide polymer derived from the colloidal red seaweed extract agar.

If proteins, oligonucleotides, or short-chain nucleic acids are being separated, then a polyacrylamide gel matrix is employed. Polyacrylamide is a polymer consisting of cross-linked repeating units of acrylic amide (acrylamide) monomers. Its pore size can be varied by changing the ratio of monomer to cross-linking agent at the time of mixing.

Polyacrylamide gel blocks can be made to have a uniform pore size, or they can be produced as so called gradient gels with a gradual decrease in pore size down the length of the block. The chief benefit of gradient gels is that they produce separations with very thin and discrete bands, allowing precise resolution of molecules that differ only slightly from one another.

As a general rule, electrophoreses employing agarose gel can be run more rapidly than those using polyacrylamide because of the larger pore size. On the other hand, the large pore size of agarose also yields reduced resolution, apparent as fuzzy and poorly-defined separation bands compared to those obtained with polyacrylamide.

DNA Gel Electrophoresis

The samples that are to be separated are loaded into wells formed at one end of the gel block in an electrophoresis apparatus such that each sample occupies a defined 'lane' across the width of the block. A power supply is connected to the apparatus with the negative electrode plugged into one end of the block and the positive electrode connected to the opposite end. In DNA electrophoresis the negative terminal is connected to the well-end of the block where the samples have been loaded. When the switch is flipped, the sample DNA will migrate from the negative to the positive end of the gel matrix. This is because DNA possesses a natural negative charge owing to the negatively charged sugar-phosphate backbone of the molecule.

As previously noted, the speed with which a molecule migrates through the matrix depends on its size and charge. Since the charge-to-mass ratio of DNA is essentially constant (owing to its structure of repeating nucleotide subunits), difference in the migration rates of DNA fragments are almost entirely due to differences in size (i.e., nucleotide base length).

The electrophoresis run is terminated once the fastest (=smallest) molecules have nearly reached the far end of the gel. Stain is then applied to the block to make visible the bands corresponding to individual molecules from the original samples. For example, ethidium bromide dye may be applied which will bind to the DNA bands and cause them to vividly fluoresce on exposure to UV light.

There are a couple of newer variations on the standard method of DNA gel electrophoresis described above. One of these, used especially for high-resolution sequencing of small DNA fragments, is to heat-denature the DNA prior to sequencing. (With larger fragments, denaturation is avoided and even reversed through a renaturing process, because long single-stranded DNA fragments tend to kink up and their tertiary structures interfere with migration through the gel.) Once denatured, the small fragments are electrophoresed through utilizing so-called 'sequencing gels,' thin polyacrylamide blocks maintained at a temperature close to the denaturing point and typically also impregnated with urea or othert denaturing compounds. Separation resolution is so powerful with this technique that DNA fragments differing in length by a single base can be discriminated.

Another variation that can enhance both speed and accuracy is capillary electrophoresis, in which the separations are run through gel-filled capillary tubes.

Electrophoretic DNA separation techniques like this are a central component of standard gene sequencing protocols described in detail in a companion page on this website. But agarose DNA electophoresis is also used as the starting point for DNA gel extraction, a technique in which bands containing viable DNA fragments are pulled off of a gel, typically through physically excising specific bands with a blade. The viable DNA is separated from the gel and by placing the excised gel piece in a length of dialysis tube to which a buffer solution is added. Electrophoresis is again applied to the gel within the tubing, forcing the DNA to migrate out of the agarose and into the buffer solution where it can be pipetted out. The result of this electroelution process is a viable sample of DNA from a specific separation band with relatively little background contamination.

Protein Gel Electrophoresis

Protein gel electrophoresis shares many procedural similarities with techniques described for DNA separation, but there are important differences as well. A key difference is that, in contrast to the polymeric repeating-unit structure of DNA, proteins vary widely in terms of their charge, shape, and structural complexity. These differences can greatly influence the rates at the molecules will migrate through a gel matrix. Some proteins will not migrate at all in their natural state when a negative to positive charge is applied. For this reason, proteins are usually denatured with detergent prior to electrophoresis. Treatment with a detergent like sodium dodecyl sulfate (SDS) denatures the proteins by disrupting non-covalent bonds, and it also coats them with a negative charge.

Importantly, SDS binds to the denatured protein at a constant ratio of one detergent anion for every two amino acid residues along the peptide chain. This applies negative charge to the sample proteins in proportion to their molecular mass, effectively giving them a comparable charge to mass ratio. In this way, the native shape and charge of the sample proteins are factored out of the equation, and migration rates in a gel electrophoresis are determined by molecule size alone.

Once the protein electrophoresis is completed, the proteins separated along the length of the gel are stained for visualization. Coomassie dye (also called 'brilliant blue') is often utilized.

Of Lanes and Ladders

A typical goal of gel electrophoresis is determination of the approximate size of unknown molecules in a sample. To facilitate this, electrophoreses usually include lanes loaded with control markers called 'ladders.' These are mixtures of DNA or protein molecules with known sizes, to which the unknown molecules can be compared after separation. Two molecules in parallel lanes that form bands the same distance from the edge of the gel are approximately the same size. Comparison of unknown bands to known markers within the ladder therefore allows meaningful size estimates for the unknown sample molecules.

Related Weblinks

Protocol Online Electrophoresis Tutorials
http://www.protocol-online.org/prot/Molecular_Biology/Electrophoresis/index.html

Arizona State University Electrophoresis Review, authored by Department of Chemistry and Biochemistry Professor Neal Woodbury
http://www.public.asu.edu/~laserweb/woodbury/classes/chm467/bioanalytical/Electrophoresis.html

Bergen County Technical Schools and Special Services, Academy for the Advancement of Science and Technology Gel Electrophoresis Tutorial Project (student-authored)
http://www.bergen.org/AAST/projects/Gel/index.html