Gel electrophoresis

By Levi Clancy for Student Reader on
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gel electrophoresis with visible bandsgel electrophoresis under uv light

Gel electrophoresis separates and visualizes molecules. To separate molecules, they are placed in agarose gel and exposed to an electric current. Larger molecules move slowly, while smaller molecules move quickly. To visualize molecules, the agarose gel is exposed to ultraviolet light. Gel electrophoresis is performed in the following steps:

  1. A mixture is loaded into wells in an agarose plate.

  2. A current is passed through the gel.

  3. Molecules migrate based on size of the molecule.

  4. Gel is exposed to an imagining molecule such as ethidium bromide.

  5. Gel is viewed under an ultraviolet light.

Notice that the leftmost lanes contain a lot of bands. Those bands are a ladder. A ladder has bands of known weight, and experimental weights can be determined by comparison to the ladder.

Gel electrophoresis techniques

ElectrophoresisUsed ForOverview

Southern Blot

DNA

DNA is extracted from cells, and is loaded into wells and run like a gel electrophoresis, in solutions that are optimized for DNA. The DNA is then transferred out of the gel onto a membrane (nitrocellulose or other), radioactive DNA is then added, and the activity is read or staining is used.

Western Blot

Protein

Protein is extracted from cells, and is loaded into wells and run like a gel electrophoresis, in solutions that are optimized for protein. The protein is then transferred out of the gel onto a membrane (nitrocellulose or other), antibodies to protein are then added, then another antibody that is conjugated with a radioactive or fluorescent molecule is added and the activity is read with X-ray or radioactivity detector or staining is used.

Northern Blot

RNA

RNA is extracted from cells, and is loaded into wells and run like a gel electrophoresis, in solutions that are optimized for RNA. The RNA is then transferred out of the gel onto a membrane (nitrocellulose or other), and probed with radioactive RNA or DNA and the activity is read or staining is used.

SDS-PAGE

Protein

SDS-PAGE electrophoresis breaks proteins into subunits, then separates the subunits based on size. Under warm conditions, SDS and β-mercaptoethanol (BME) are added. SDS coats subunits with an anionic charge, then a current is applied to the gel. Smaller subunits migrate through the gel's pores fastest, thereby moving farther. Like with other forms of electrophoresis, a solution containing subunits of known sizes (known as a ladder) is used as a reference point. Bands of subunits can be cut from the gel and purified. Perturbation and handling of the gel means this technique is not completely accurate, but it is still very effective.

Zig-Zagging

Genome

A technique was developed to perform gel electrophoresis of whole genomes, which shear very easily when loading the wells and which travel very slowly through agarose gel due to their size.

  1. Use SDS to lyse cells directly in agarose wells to prevent shearing of DNA during loading.

  2. Change electrophoretic direction (90s ↓; 90s →) to pull DNA through agarose in a zig-zag.