Gel Electrophoresis — how it works and what it can be used for
Gel electrophoresis is an important laboratory technique used to separate charged molecules such as DNA through the use of a current. Electrophoresis was first developed by Arne Tiselius in the 1930s — since then, countless variations have been made on Tiselius’ original method, resulting in, among others, the gel electrophoresis that is so widely used today.
Set Up
Gel electrophoresis takes place on a tray containing a sticky, porous gel (most commonly polyacrylamide or agarose) as well as a buffer which is needed as it ensures the charge of the tested molecules doesn’t change. This is joined up to a power supply and, when this is turned on, electrons are able to flow through the gel from the cathode (negative electrode) to the anode (positive electrode). By the cathode there are several sample wells which is where the molecules to be separated must be placed before the current is switched on.
How it works
One important feature of DNA is its phosphate backbone: when DNA is dissolved, the positive end of the water molecules are able to pull a H+ ion off each phosphate group, causing the DNA molecule to take on a negative charge. Since the charge of DNA is directly proportional to the molecule’s length, all DNA molecules have the same ratio of charge to mass: gel electrophoresis of DNA therefore will separate it on the basis of size only.
When the power supply is turned on, the negatively charged DNA fragments are attracted to the positively charged anode on the other side of the tray which causes them to move through the gel towards it. However, because the gel is porous, a ‘sieving’ effect is created: smaller DNA molecules can pass through the pores easier than larger ones, and will therefore have moved further across the gel in the same time frame.
Analysing results
After a set amount of time has passed, the power supply will be turned off and a DNA–binding dye will be added to the tray. When the tray is placed under UV light, it will reveal distinct bands of colour (see below), each band corresponding to a certain length of DNA. Often, one of the lanes on the tray will be reserved for a so–called DNA ladder, which contains known lengths of DNA to act as a reference for the other DNA fragments: given the reliability of electrophoresis, we can accurately interpolate the approximate length of each DNA fragment based on the distance it has travelled. Bands closer to the sampling well correspond to longer DNA molecules while those further away are shorter in length. Electrophoresis may be followed by further analytical techniques such as Southern blotting for further characterisation of DNA fragments.
Electrophoresis for proteins
Proteins take on complex tertiary structures and can have variable charges depending on the exact amino acids present. This, alongside molecular mass, can affect the rate at which each protein molecule moves through the gel and therefore confound results. This issue is generally resolved by first denaturing the proteins in a detergent such as sodium dodecyl sulfate which unfolds the proteins and coats them in a negative charge, making them act similarly to nucleic acids.
An alternative to this is 2d electrophoresis, which was developed in 1975. Here, the proteins are first separated in one dimension based on their charge, after which are separated in a second dimension based on their molecular mass. Since it is unlikely two proteins will be the same in both of these properties, it is a more effective way of separating proteins than 1d electrophoresis.
How is it useful?
Gel electrophoresis is generally used for analytical purposes and is often used following PCR to check that a desired section of DNA (which will have a set length) has been amplified. It is also helpful in DNA cloning since it can show whether a set gene has been successfully inserted into a bacterial plasmid to mass-produce a desired protein. As a separation technique, however, electrophoresis can also be useful in isolating individual kinds of molecules from what may otherwise be a mixture of different DNA fragments or proteins, and many further laboratory techniques, like the previously mentioned blots, rely on gel electrophoresis as a first step in determining the quantity and/or identity of various macromolecules.
