Gel Electrophoresis

Gel electrophoresis refers to using a gel as an anticonvective medium and or sieving medium during electrophoresis. Gel electrophresis is most commonly used for separation of biological macromolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein; however, gel electrophoresis can be used for separation of nanoparticles. Electrophoresis refers to the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied. 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 spectrometryRFLPPCRcloningDNA sequencing, or Southern blotting for further characterization.


Types of Electrophoresis

Electrophoresis is one of the most popular ways to analyze macromolecules such as proteins, DNA and RNA. There are several types of electrophoresis, but the concepts are similar. The machine has an anode (positive charge) and a cathode (negative charge). Negative ions move toward the anode, and positive-charged ions move towards the cathode. The rate and distance traveled by these molecules help scientists classify and study different biomolecules.
  1. SDS-PAGE

    • SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) is a common form of electrophoresis for analyzing proteins. The SDS part of the name is a protein denaturing detergent that causes the molecule to unfold. The detergent binds to the polypeptide in a 1:1 ratio with each segment of the protein to give it a charge. The protein polypeptides move through the gel at different rates depending on mass, allowing researchers to study proteins based on size.

    Agarose Gels

    • Agarose gels are an electrophoresis method to separate RNA and DNA molecules. Like SDS-PAGE,this separates the molecules based on charge and size. DNA molecules are negatively charged, so they move through the gel quickly depending on size. Smaller DNA fragments move more quickly than larger ones due to friction resistance.

    Electrofocusing

    • Electrofocusing takes advantage of charge and pH values of proteins. A container is filled with a gel solution that has an increasing pH gradient. The amino acids that form polypeptides have different acidic or basic charges. The protein travels through the gel, obtaining or losing protons depending on its charge. As the protein particle moves through the gel, it eventually becomes neutral and gets stuck in an isoelectric position.

    Capillary

    • Capillary electrophoresis is a method similar to SDS-PAGE. It separates molecules based on their charge and mass. Molecules are placed in rows called capillaries filled with conductive, electrolyte fluid. The analytes move in a speed relative to their charge and mass. This method is an older technique introduced in the 1960s. SDS-PAGE is usually preferred in labs.

    Native Gels

    • Native gels are similar to SDS-PAGE, except the detergent (SDS) is not used to denature proteins. Native gels are only able to separate proteins up to 2,000 kDa in size. Because the proteins are left folded, the dyes used are also different than SDS-PAGE. Researchers are able to view proteins based on mass and charge using enzymes that catalyze protein-specific reactions.

      http://www.ehow.com/about_5047959_types-electrophoresis.html




Principles of Electrophoresis


Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.

When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.

Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.

The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).

Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.

Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html

Agarose Gel Electrophoresis of DNA


Preparing and Running Standard Agarose DNA Gels

The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:

  • An electrophoresis chamber and power supply
  • Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
  • Sample combs, around which molten agarose is poured to form sample wells in the gel.
  • Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
  • Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
  • Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling.
  • Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light.

To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.

After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.

The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.

When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis.

Migration of DNA Fragments in Agarose

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.

Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.

Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

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