Gel Electrophoresis

Gel Electrophoresis Definition

Gel Electrophoresis is a procedure used in molecular biology to separate and identify molecules (such as DNA, RNA, protein, complexes) by size. The separation of these molecules is achieved by placing them in a gel made up of small pores and setting an electric field across the gel. The molecules will move based on their inherent electric charge (i.e., negatively charged molecules move away from the negative pole) and smaller molecules will move faster than larger molecules; thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size; the electrophoresis works to move the particles, using their inherent electric charge, through the sieve.

Purpose of Gel Electrophoresis

The purpose of gel electrophoresis is to visualize, identify and distinguish molecules that have been processed by a previous method such as PCR, enzymatic digestion or an experimental condition. Often, mixtures of nucleic acids or proteins that are collected from a previous experiment/method are run through gel electrophoresis to determine identity or differentiate molecules.

Types of Gel Electrophoresis

There are two types of gel electrophoresis: native and denaturing. A native gel electrophoresis usually attempts to keep RNA or protein in its native structure while running it through the gel. A denaturing gel electrophoresis attempts to reduce the RNA or protein into its most linear structure before or during gel electrophoresis. The denaturation of the RNA or protein is accomplished by adding a reducing agent to the sample, gel and/or buffer. The reducing agent separates bonds within the RNA or protein molecule and thereby reduces its secondary structure. The secondary structure of a protein or RNA will influence, in a non-linear manner, how fast it migrates through a gel. A denatured, linear form of RNA or protein, however, will migrate proportionally to its linear size (base pairs or kilo Daltons). Denaturing gel electrophoresis is often more accurate for size identification, whereas native gel electrophoresis is usually used to identify protein complexes.

Examples of Gel Electrophoresis

  • TAE Agarose Gel Electrophoresis is most commonly used for DNA.
  • TBE and Denaturing PAGE (polyacrylamide gel electrophoresis) are common for RNA separation.
  • SDS PAGE is a denaturing gel electrophoresis commonly used for protein identification and separation.

Gel Electrophoresis Steps

The broad steps involved in a common DNA gel electrophoresis protocol:

1. Preparing the samples for running

The DNA is isolated and preprocessed (e.g. PCR, enzymatic digestion) and made up in solution with some basic blue dye to help visualize the movement of the sample through the gel.

2. An agarose TAE gel solution is prepared

TAE buffer provides a source of ions for setting up the electric field during electrophoresis. A weight to volume concentration of agarose in TAE buffer is used to prepare the solution. For example, if a 1% agarose gel is required, 1g of agarose is added to 100mL of TAE. The agarose percentage used is determined by how big or small the DNA is expected to be. If one is looking at separating a pool of smaller size DNA bands (<500bp), a higher percentage agarose gel (>1%) is prepared. The higher percentage agarose creates a denser sieve to increase separation of small DNA length differences. The agarose-TAE solution is heated to dissolve the agarose.

3. Casting the gel

The agarose TAE solution is poured into a casting tray that, once the gel solution has cooled down and solidified, creates a gel slab with a row of wells at the top.

4. Setting up the electrophoresis chamber

The solid gel is placed into a chamber filled with TAE buffer. The gel is positioned so that the chamber wells are closest to the negative electrode of the chamber.

5. Loading the gel

The gel chamber wells are loaded with the DNA samples and usually a DNA ladder is also loaded as reference for sizes.

6. Electrophoresis

The negative and positive leads are connected to the chamber and to a power supply where voltage is set. Turning on the power supply sets up the electric field and the negatively charged DNA samples will start to migrate through the gel and away from the negative electrode towards the positive.

7. Stopping electrophoresis and visualizing the DNA

Once the blue dye in the DNA samples has migrated through the gel far enough, the power supply is turned off and the gel is removed and placed into an ethidium bromide solution. Ethidium bromide intercalates between DNA and is visible in UV light. Sometimes ethidium bromide is added directly to the agarose gel solution in step 2. The ethidium bromide stained gel is then exposed to UV light and a picture is taken. DNA bands are visualized in from each lane corresponding to a chamber well. The DNA ladder that was loaded is also visualized and the length of the DNA bands can be estimated. An example is given in the figure below.

Gel electrophoresis

Gel electrophoresis

Quiz

1. The gel in gel electrophoresis is analogous to what apparatus?
A. Power supply
B. Sieve
C. Current generator
D. A ladder

Answer to Question #1
B is correct. The gel serves to separate large and small molecules with its porous meshwork, through which smaller molecules can travel faster than larger molecules.

2. What does gel electrophoresis accomplish?
A. Adds electric charge to molecules
B. Switches the electric charge of molecules
C. Adds electric charge to gels
D. Separates molecules by their size

Answer to Question #2
D is correct.

3. Identify a reason to run a native versus denaturing gel electrophoresis.
A. For accurate protein size identification
B. For identifying a protein bound to another protein
C. To separate two pieces of DNA
D. To get two proteins to bind to one another

Answer to Question #3
B is correct. For identifying a protein bound to another protein. The main purpose of gel electrophoresis is to identify molecules. The purpose of native gel electrophoresis is to keep protein structure intact while having it migrate through the gel. The intact structure allows the protein to maintain its bound or complex formation with other proteins. A denaturing gel electrophoresis would technically be more accurate for identifying the size of proteins.

References

  • Green, M.R and Sambrook J. (2012). Molecular Cloning A Laboratory Manual, 4th ed. Ch.2. CSHLP: New York. ISBN: 1936113422
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