Julian's Science Experiments
  • Famous Experiments and Inventions
  • The Scientific Method
  • Home Biochemistry Experiments Biochemistry Science Fair Projects Resources Biology Jokes Warning!
       

    Gel Electrophoresis
    K-12 Experiments & Background Information
    For Science Labs, Lesson Plans, Class Activities & Science Fair Projects
    For High School Students & Teachers







    Gel Electrophoresis Experiments

    Gel Electrophoresis Background Information

    Definitions

    Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix.

    Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss, who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.

    Basics

    DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

    Separation: The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules and macromolecular complexes.

    "Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively charged or toward the cathode if positively charged.

    Visualization: After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

    If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

    Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

    Applications: Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

    Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications.

    Nucleic acids: In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.

    Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size or, for cyclic fragments, their radius of gyration. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

    Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift affinity electrophoresis.

    Proteins: SDS-PAGE autoradiography - The indicated proteins are present in different concentrations in the two samples.Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacryl amide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

    History:

    • 1930s - first reports of the use of sucrose for gel electrophoresis
    • 1955 - introduction of starch gels, mediocre separation
    • 1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of parameters such as pore size and stability
    • 1964 - disc gel electrophoresis (Ornstein and Davis)
    • 1969 - introduction of denaturing agents especially SDS separation of protein subunit (Weber and Osborn)
    • 1970 - Laemmli separated 28 components of T4 phage using a stacking gel and SDS
    • 1975 - 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis
    • 1977 - sequencing gels
    • late 1970s - agarose gels
    • 1983 - pulsed field gel electrophoresis enables separation of large DNA molecules
    • 1983 - introduction of capillary electrophoresis

    Topics of Interest

    Native Gel Electrophoresis is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio.

    Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels.

    Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones.

    Pulsed field gel electrophoresis: The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 120 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely large bands (up to around 2Mb), switching-interval ramps can be used that increases the pulse time for each direction over the course of a number of hours--take, for instance, increasing the pulse linearly from 10 seconds at 0 hours to 60 seconds at 18 hours.

    Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) are forms of electrophoresis where there is a temperature or chemical gradient across the gel. TGGE and DGGE are useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins.

    Difference gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three different protein samples can be labeled with fluorescent dyes (for example Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis. After the gel electrophoresis, the gel is scanned with the excitation wavelength of each dye one after the other. This technique is used to see changes in protein abundance (for example, between a sample of a healthy person and a sample of a diseased person).

    Source: Wikipedia (All text is available under the terms of the GNU Free Documentation License and Creative Commons Attribution-ShareAlike License.)

    Useful Links
    Biochemistry Resources
    Biochemistry and Cell Biology Science Fair Projects and Experiments
    General Science Fair Projects Resources
    Biology / Biochemistry Science Fair Projects Books

                  





    My Dog Kelly

    Follow Us On:
           

    Privacy Policy - Site Map - About Us - Letters to the Editor

    Comments and inquiries could be addressed to:
    webmaster@julianTrubin.com


    Last updated: June 2013
    Copyright © 2003-2013 Julian Rubin