Agarose gel electrophoresis is commonly used for the analysis and separation of biomolecules, especially nucleic acid fragments based on their size. It can also be used for separation of high molecular weight protein or protein complexes. Low molecular weight biomolecules such as most proteins (except very high molecular weight proteins) and small nucleic acid fragments can not be effectively separated by agarose gel electrophoresis.
Agarose gel electrophoresis has to main components:
- Agarose gel
- Electric field
Agarose gel, a porous matrix, acts as a sieve through which molecules can move. Electric field is the force that forces molecules to move through an agarose gel matrix. The molecules must have a net charge, positive or negative, in order to move in an electric field. A neutral molecule will not move in an electric field.
When a molecule moves through an agarose gel matrix, the gel matrix poses hindrance to its movement. This results in retardation in the migration speed and the extent of retardation depends on the size of the molecule. Consequently, larger molecules will move slower than smaller molecules, which ultimately be seen as a separate band on agarose gel. For example, if you have digested plasmid DNA which contains DNA fragments of sizes 1000 bp and 4000 bp, they will run on agarose gel at two different positions as discrete bands: 1000 bp fragments will be running faster than 4000 bp fragments.
If the molecules are too small (as compared to pore size of the agarose matrix), they will freely move through agarose gel and if the molecules are too big, they will have difficulties entering the agarose gel. In both cases, there will not be any effective separation. This sets the size limit of the molecules to be separated by agarose gel electrophoresis. Agarose gel is usually used to separate DNA fragments between 100 bp – 30,000 bp (2% – 0.5% agarose gel). DNA fragments lower than 100 bp can be separated by polyacrylamide gel electrophoresis (smaller pore size than agarose gel) and for larger fragments, pulse-field gel electrophoresis can be used.
The pore size of agarose gel can be controlled by changing the agarose percent. Pore size is inversely proportional to the percentage (concentration) of agarose in gel. Higher percent agarose gels have a smaller pore size. This provides an opportunity to choose an appropriate percent agarose gel for the separation of different size molecules. There is also a limit in controlling agarose gel matrix pore size. Since a very low percentage agarose gel is very difficult to handle as they are very delicate and can be broken while handling, agarose gel with agarose concentration lower than 0.5% is usually not used. Higher percentage agarose gels are also difficult to cast as the molten agarose is very viscous and solidifies quickly even when it is hot. Very hot molten agarose solution can also damage and destroy your casting tray. Usually 2% agarose gel can be casted without much difficulties.
Electric field is required to force charged molecules to move through an agarose matrix. Ionic medium is required for the conduction of electricity, therefore, an electrophoresis buffer is used for this purpose. Since electricity has to be passed through agarose gel, gel is also prepared by melting agarose in the electrophoresis buffer.
In order to move under an electric field, the molecules must have net charge at pH of the electrophoretic buffer. If a molecule has a net negative charge, it will move towards a positive electrode, and if it has net positive charge it will move towards a negative electrode. DNA has net negative charge due to its phosphate backbone at neutral or slightly basic pH (the pH of most electrophoresis buffers used for DNA agarose gel electrophoresis), therefore, moves towards positive electrode. Since the DNA mass/charge ratio is the same for all different size fragments, the separation happens only based on size of DNA.