Gel electrophoresis
Gel electrophoresis is used to separate macromolecules like DNA, RNA and proteins. DNA fragments are separated according to their size. Proteins can be separated according to their size and their charge (different proteins have different charges).
How are DNA fragments separated using gel electrophoresis?
Gel electrophoresis: Making the gel
Tasha is a Year 13 student who learnt how to make a gel at Genesis R&D in Auckland.
The gel can be made using a gelling agent like agarose. The agarose is mixed with buffer and heated so that it dissolves. Ethidium bromide may be added. This is a chemical that binds to DNA and fluoresces (shines up) under UV light so that the position of the DNA in the gel can be visualised.
Once the ethidium bromide is added, the agarose solution is poured into a mould and allowed to set. 'Combs' are placed into the gel to create moulded wells where the DNA / protein solution can be added.
Gel electrophoresis: Making the gel
Tasha is a Year 13 student who learnt how to make a gel at Genesis R&D in Auckland.
The gel can be made using a gelling agent like agarose. The agarose is mixed with buffer and heated so that it dissolves. Ethidium bromide may be added. This is a chemical that binds to DNA and fluoresces (shines up) under UV light so that the position of the DNA in the gel can be visualised.
Once the ethidium bromide is added, the agarose solution is poured into a mould and allowed to set. 'Combs' are placed into the gel to create moulded wells where the DNA / protein solution can be added.
A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules.
The result is a series of ‘bands’, with each band containing DNA molecules of a particular size. The bands furthest from the start of the gel contain the smallest fragments of DNA. The bands closest to the start of the gel contain the largest DNA fragments.
Gel electrophoresis: Loading and running the gel
Caleb is a Year 13 student who learnt how to load and run a gel at Genesis R&D in Auckland. He shows us how it's done.
Gel electrophoresis: Loading and running the gel
Caleb is a Year 13 student who learnt how to load and run a gel at Genesis R&D in Auckland. He shows us how it's done.
Gel electrophoresis: Visualising and interpreting the results
A chemical called ethidium bromide had been added to the gel. It binds to the DNA fragments in the gel. It also fluoresces, or lights up, under UV light. This means that the DNA fragments can be seen in UV light. The DNA fragments shine up as 'bands'. Each band contains DNA fragments of the same size (because they have travelled the same distance through the gel).
A 'reference ladder' can also be run in the gel. This contains a mixture of DNA fragments of known size. Comparing the bands in your DNA sample with the bands in the reference ladder allows you to work out how big the DNA fragments are in a particular band. DNA size is measured in base pairs (bp), or kilo-base pairs. (Remember that DNA is a chain of nucleotides, each nucleotide consisting of a phosphate group, a sugar, and a base. DNA is also double stranded. One base pair therefore represents one pair of nucleotides in the double stranded DNA molecule. A molecule that is 200bp long would consist of 200 pairs of nucleotides).
Gel electrophoresis: Visualising and interpreting the results
A chemical called ethidium bromide had been added to the gel. It binds to the DNA fragments in the gel. It also fluoresces, or lights up, under UV light. This means that the DNA fragments can be seen in UV light. The DNA fragments shine up as 'bands'. Each band contains DNA fragments of the same size (because they have travelled the same distance through the gel).
A 'reference ladder' can also be run in the gel. This contains a mixture of DNA fragments of known size. Comparing the bands in your DNA sample with the bands in the reference ladder allows you to work out how big the DNA fragments are in a particular band. DNA size is measured in base pairs (bp), or kilo-base pairs. (Remember that DNA is a chain of nucleotides, each nucleotide consisting of a phosphate group, a sugar, and a base. DNA is also double stranded. One base pair therefore represents one pair of nucleotides in the double stranded DNA molecule. A molecule that is 200bp long would consist of 200 pairs of nucleotides).
When is gel electrophoresis used to separate DNA fragments?
Gel electrophoresis can be used for a range of purposes, for example:
To get a DNA fingerprint for forensic purposes
To get a DNA fingerprint for paternity testing
To get a DNA fingerprint so that you can look for evolutionary relationships among organisms
To check a PCR reaction. See the video clip: Using gel electrophoresis to check a PCR reaction
To test for genes associated with a particular disease. Find out more in the article, Gel electrophoresis can be used to find genes associated with a disease.
When is gel electrophoresis used to separate proteins?
Thanks to TV shows like CSI, many people are familiar with the use of gel electrophoresis to separate macromolecules like DNA. However, gel electrophoresis can also be used to separate out proteins.
Different proteins have different sizes, mainly due to the number of amino acid building blocks in their structure. Chemical modifications attached to the protein also affect its size. Different proteins also have different charges. This can result from both the types of amino acid used to construct them, as well as the types of modifications attached to them.
Different types of electrophoresis gels are used to provide different types of information. The type of gel you choose therefore depends on the type of question you are asking.
Size Separation
Protein Electrophoresis
SDS-PAGE gel electrophoresis separate proteins from the blood of different shark species.
Typically, gels made from polyacrylamide are used to separate proteins on the basis their different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in order to unravel the protein. SDS is a detergent that gives all the proteins the same overall negative charge so that when an electric current is applied to the gel, separation is only due to the size of the protein. This technique is called SDS-PAGE (SDS-Polyacrylamide gel electrophoresis).
Small protein molecules move more quickly through the gel than larger proteins, resulting in a series of ‘bands’. Each band contains a protein of a particular size. These can be compared with standards of known sizes.
An SDS-PAGE gel has been used to separate proteins on the basis of size. The samples are the blood of various shark species. The first lane contains markers of known sizes. Large proteins are at the top of the gel and small proteins are at the bottom.
This technique might be used for many purposes, including purifying a particular protein, for example to isolate an enzyme for the food industry.
Charge and pH separation
Isoelectric focusing (IEF) and agarose gel electrophoresis are two ways that proteins can be separated by their different electrical charges. Unlike SDS-PAGE, the proteins are usually kept in their native (folded) state. The type of gel that is used, and the solution around the gel, are also different.
In agarose gel electrophoresis, proteins are loaded in the middle of the well. Those with a strong negative charge move fastest towards the positive side of the gel, whereas positively charged proteins move in the opposite direction.
This technique might be used to separate proteins that have the same molecular weight but different charges, or when size is not important (e.g. to look at changes in the presence of different protein during the development of a disease).
Two-dimensional electrophoresis
These days, charge (IEF) and size (SDS-PAGE) separation are often employed together in two-dimensional electrophoresis, where charge separation is first used, and then these separated proteins are separated on the basis on size.
This is a very effective method for identifying a particular protein from a tissue that may contain thousands of proteins and where there may only be small differences between control and treated samples (e.g. to look for a protein involved in resistance to insect predation in plants).
