Gel electrophoresis

File:Electrophoresis - Filling 1D gel wells with proteins mixture.jpg

Gel electrophoresis machine. The mixture is placed in the well. The gel is held by plates. The electric field causes the gel to have a positive and negative side on opposite ends

Gel electrophoresis is a technique used to separate mixtures like DNA and proteins. The separation is based on how positively or how negatively charged a molecule is, and its size. Gel electrophoresis uses a gel (like gelatin) and an electric field is put through the gel.

The word electrophoresis comes from –electro, because an electric field is used, and –phoresis, which means movement.

Gel

The gel is made up of large and branched molecules called polymers. The amount of branches in the gel determines how easily molecules can squeeze through, depending on their size.

If there are a lot of branches, small molecules will be able to move through easily while big molecules will move through slowly or not at all. If there are few branches, both big and small molecules can move through more easily.

Charge on the molecule

File:AgarosegelUV.jpg

Block of gel seen under ultraviolet (UV) lamp. The bands are colored red

If a large molecule has a big charge, its attraction to the opposite charge is also large. But, since the molecule is large, it will have a difficult time moving through the thick gel. In gel electrophoresis, large molecules are going to move slower.

A small charged molecule will move through the gel more easily. Shorter molecules move faster and move further than longer ones because shorter molecules get through the pores of the gel more easily. This phenomenon is called sieving.

If the molecule does not have any charge, it will not move.

Visualization

After the gel has been run by applying the electric field, you can look to see where the molecules have moved to. To do this, different stains can be applied to the gel: this allows the molecules to be seen. The stain makes the places where the molecules moved to appear as colored bands. Stains that are used to look at proteins, such as Coomassie blue, can often been seen by the human eye under normal light. Stains that are used to look at DNA, such as ethidium bromide and GelGreen, can only be seen with the help of an ultraviolet lamp.

DNA separation

Gel electrophoresis is the most commonly used technique to study DNA. DNA is a very large molecule that contains genetic information. DNA can be broken down to smaller pieces of different sizes and these pieces are then separated using gel electrophoresis. DNA always has a negative charge, and moves towards the anode.

Proteins are large and complex molecules made of amino acids. Proteins can be studied by gel electrophoresis in two ways. One way is to take a mixture of proteins and separate them in the gel. The other way is to break down a single protein into smaller pieces. The smaller pieces can then be separated in the gel. Proteins can be positively or negatively charged. To separate proteins by size only, protein mixtures can be coated with a chemical called sodium dodecyl sulfate (SDS) to give all proteins a negative charge before putting the mixture into the gel.

Medicine

In medicine, there is a special type of electrophoresis called iontophoresis. Iontophoresis uses the same idea of gel electrophoresis to deliver drugs into the human body through the skin without using needles to inject the drug.

Gel Electrophoresis Media

• Gel Electrophoresis.svg

  The image above shows how small DNA fragments will migrate through agarose quickly but large size DNA fragments move more slowly during electrophoresis. The graph to the right shows the nonlinear relationship between the size of the DNA fragment and the distance migrated.

• Gel Electrophoresis in DNA Fingerprinting.svg

  Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples. DNA is extracted. * Isolation and amplification of DNA. * DNA added to the gel wells. * Electric current applied to the gel. * DNA bands are separated by size. * DNA bands are stained. *

• SDS-PAGE Electrophoresis.png

  Overview of gel electrophoresis.

• Gel electrophoresis insert comb.jpg

  Inserting the gel comb in an agarose gel electrophoresis chamber

• Glucose-6-Phosphate Dehydrogenase activity stain.jpg

  Specific enzyme-linked staining: Glucose-6-Phosphate Dehydrogenase isoenzymes in Plasmodium falciparum infected Red blood cells

• Pcr gel.png

  An agarose gel of a PCR product compared to a DNA ladder.

• SDSPAGE.png

  SDS-PAGE autoradiography – The indicated proteins are present in different concentrations in the two samples.