An Introduction to Gel Electrophoresis – DNA, RNA & Proteins
The term gel electrophoresis refers to a technique used for separation and analysis of DNA, RNA , and proteins based on their size and charge. The suffix phoresis means “migration” or “movement”, while the prefix electro indicates the use of electricity as a mean to separate molecules.
In a typical gel electrophoresis system, an electric field is generated by connecting the two opposite ends of a gel tank to a power supply. One end will become positively charged, while the opposite end negatively. This electric current will cause DNA, RNA and protein fragments to migrate along the gel.
DNA gel electrophoresis
DNA gel electrophoresis is incorporated into various techniques as a preparative technique in molecular biology such as PCR, DNA sequencing, genome mapping and Southern blotting. A gel electrophoresis can run both horizontally and vertically, however, standard DNA and RNA gels run horizontally.
DNA and RNA are negatively charged molecules, once they are loaded into the gel from the negative end of the gel and exposed to an electric field, they migrate through the gel pores towards the positively charged end of the gel.
Gels for DNA or RNA separation are often made out of a polysaccharide called agarose that makes up the gel matrix. When the agarose is heated in a buffer and cooled down, it will form a solid gel. The gel matrix will act as a sieve; the greater the agarose concentration, the smaller the pores created in the gel matrix, and the more difficult it is for large linear DNA molecules to move through the matrix.
Different-size fragments will migrate at different rates, therefore allowing the researcher to identify the fragment of interest among a mixed sample. After electrophoresis, the gel can be visualized under UV lights thanks to the addition of DNA intercaletor, such as Ethidium Bromide to the running gel.
Preparing the gel
Agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted (a microwave oven is generally used at this step).
Electrophoresis buffers are used to provide ions that carry a current and to maintain the pH at a relatively constant value. The most common electrophoresis buffers for nucleic acids are Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE). In addition, 0.2-0.5 μg/mL of Ethidium bromide will be added to the buffer.
Gel % varies depends on the size of the fragment
|Percent Agarose Gel (w/v)||DNA Size Resolution (kb = 1000)|
|0.5%||1 kb to 30 kb|
|0.7%||800 bp to 12 kb|
|1.0%||500 bp to 10 kb|
|1.2%||400 bp to 7 kb|
|1.5%||200 bp to 3 kb|
|2.0%||50 bp to 2 kb|
Table 1: Correct Agarose Gel Concentration for Resolving DNA fragments
|Polyacrylamide gel||Proteins and nucleic acids|
|Agarose gel||Nucleic acids and nucleoproteins, etc.|
Table 2: Type of Gels for Resolving DNA/RNA or protein fragments
How do DNA fragments move through the gel?
Visualizing the DNA fragments
When the run has ended, the gel is placed under under a UV light source. Thanks to the addition of Ethidium bromide, intercaeletor-bound DNA fragments will glow and become visible as distinct bands along the gel’s length. The exact size of each DNA band is detected by adding a molecular marker made up of several bands of known size.
The molecular weight of linear double stranded DNA run in agarose gel is estimated by the addition of DNA molecular markers often referred to as DNA Ladders. These ladders contain precisely quantified and measured linear double stranded fragments of DNA pre-mixed with loading dye making them ready to use.
At Expedeon we offer three DNA ladders:
- The 50bp PLUS ladders (from 50bp up to 1000bp)
- The 100bp ladders (from 0.1kb to 3kb bp)
- The 1kb PLUS (from 0.1kb to 10kb)
Proteins are not negatively charged and as such cannot be separated by the application of an electric field. To overcome this issue, detergent sodium dodecyl sulfate is added to the protein solution in order to separate proteins using gel electrophoresis. This treatment causes the proteins to unfold into a linear shape while covering them with a negatively charged coat. This negative coat allows proteins to migrate towards the positively charged end of a gel and therefore be separated by molecular weight.
The purpose of using stain is to have a clear background with blue-stained protein bands. Some premade and traditional home-made protein Coomassie R-250 stains can take 3 hours or more to fully stain gels and it is necessary to fix and wash the gel before adding the stain.
Check out Expedeon’s RunBlue Prestained Molecular Weight Markers.
To overcome this issue, we offer easy to use Expedeon’s InstantBlue stain which is a ready to use Coomassie protein stain for polyacrylamide gels. Its unique mechanism of action stains proteins in 15 minutes while leaving a clear background, eliminating the need to fix, wash or destain.
While DNA fragments can be immediately visualized under UV lights, protein fragments need to be labeled with target-specific antibodies, a process known as immunostaining. This reaction cannot happen on a gel substrate; proteins must be transferred onto a membrane (mostly PVDF or Nitrocellulose) then blotted with a solution containing an antibody that will specifically recognize and bind the protein of interest.
The target-antibody conjugation can be visualized by the addition of a label to the chosen antibody. This process can be now easily performed in-house with one of our Lightning-Link kits.
If you want to know more about antibody labeling, download our guide.
A gel is composed of polyacrylamide or agarose. RunBlue Bis-Tris Protein Gels have been developed by Expedeon to provide an alternative to NuPAGE®Bis-Tris gels. It also has benefits such as comb/strip free design and tear proof composition.
Why not check our other products that can assist you further in your gel electrophoresis experiment? To find out more, please click on the links below
Products and accessories
View our range of buffers, prestained molecular weight markers and protein stains here.
We also offer a range of electrophoresis equipment for DNA and protein electrophoresis:
If you have anymore questions regarding our range of products or need technical support, please dont hesitate to get in touch.Click to read more