Have you been searching for a method to separate a mixture of proteins and DNA? Or maybe you’re searching for a way to separate a mixture of different proteins or want to know how to separate a mixture of different DNA strands? If so, you’ve found what you have been looking for. After reading this article, you will understand how gel electrophoresis works and learn about the two most common types used to separate proteins and DNA- agarose gel electrophoresis and polyacrylamide gel electrophoresis SDS-PAGE. If you work with proteins and/or DNA, you’ll find this information very useful because these are the most widely used techniques for DNA and protein separation.

What is Gel Electrophoresis?

Gel Electrophoresis is a technique used to separate DNA, RNA and proteins, based on their molecular size. To begin the process, the sample you wish to separate is added to a porous gel, where its biological molecules are forced through the gels pores in the presence of an electric field. The molecules move through the pores at a rate that is inversely proportional to the size. Therefore, smaller molecules will travel through the gel, faster and longer, than larger molecules will.

The electric field is utilized so that the there is a separation of charges at either end of the device, i.e. one end is positively charged, and the other is negatively charged. This causes the negative charges on the surface of the DNA to move towards the positive end of the gel. Proteins are not charged so they do not migrate. This allows for an effective separation of nucleic-based molecules and proteins. Also, as smaller DNA chains will migrate faster, you can also separate short and long chain DNA molecules. DNA markers with known lengths can also be added into the matrix allowing for an easy deduction of DNA lengths, as they will migrate to a given region.

If you would like to separate the proteins from the mixture, then the surface of the protein needs to be modified with a surfactant molecule, such as sodium dodecyl sulfate. The surfactant molecule breaks down the intermolecular forces in the protein, causing an unfolding in the tertiary structure of the protein making them linear, whilst coating them with a negative charge. This allows them to migrate to the positive section of the gel. Upon separation, bands of molecules (which have been stained by a fluorescent or radioactive dye) with different sizes can be detected. Ethidium bromide is the most common choice of stain.

There are different types of Gel Electrophoresis available dependent upon your applications. The main differing factor is the type of gel used.

Agarose Gel Electrophoresis

Agarose gel Electrophoresis is a technique primarily used to separate and detect DNA molecules. After separation the molecules can be seen under UV-light after being stained. It uses a cast agarose gel.

An agarose gel has a three-dimensional matrix consisting of helical agarose molecules that are aggregated together to form pores. Agarose is a strong gel with large pores and a high melting temperature, but can be chemically modified to provide a lower melting or gelling point, if required. They are also resistant to UV-light degradation, and are easy to cast. A strong gel concentration is around 1% agarose gel (ranges are between 0.7-2%, for large and small DNA molecules, respectively). At this concentration the pore sizes range between 100 and 250 nm. Any lower than this, then the gel becomes fragile. Any higher, and the pores become too small for effective separation and migration. Agarose gels are typically used to separate DNA molecules which consist of 50 to 25,000 base pairs, due to a low resolving power. It has a limit of 750,000 base pairs. For proteins, the optimal weight is 200 kDa.

There are many factors that can affect how the DNA molecules flow through the gel matrix, including: the size of DNA molecule, agarose concentration, DNA conformation, voltage applied, presence of ethidium bromide, type of agarose and an electrophoresis buffer (generally an EDTA based molecule).

However, some of the polymers within an agarose gel are charged and can cause a water flux displacement, opposite to that of the DNA movement. This can cause problems with the DNA movement, which can cause a blurring of the DNA bands. Overloading of DNA samples and impurities can also affect the movement of the DNA molecules.

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) uses a stacked gel (two gels separated by a buffer) and can be used with both proteins and DNA, but is more common with proteins. The common denaturant for DNA molecules is urea. For proteins it is sodium dodecyl sulfate (SDS), which leads to the common name of SDS-PAGE, for the separation of proteins. Samples are prepared by a variety of techniques including blending, homgenizing, sonicating, high pressures, filtration and centrifugation.

The polyacrylamide gel consists of many constituents. The complex mixture consists of acrylamide, bisacrylamide, denaturant and a pH buffer (bis, tris or imidazole based molecule). The acrylamide and bisacrylamide concentrations are normally in the region of 5-25%. Lower concentrations are better for high weight molecular proteins, as the bisacrylamide molecules cross-link the acrylamide molecules to form pores. This makes the gel highly tuneable. So the lower the concentration, the larger the pores, and vice versa. PAGE-SDS is used to separate proteins with a molecular weight of 5 kDa to 2000 kDa.

Once proteins or DNA have been separated, they can be analyzed in blot tests. The are four types of blot test; the Northern, Southern, Eastern and Western, which are used to analyse the gene expression in DNA, detection of a specific DNA sequence, protein translational modifications and detection of a specific protein, respectively.