Proteomics/Protein Separations- Electrophoresis/What is Electrophoresis?
- What is Electrophoresis?
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Gel Electrophoresis is a separation technique that is used to separate macromolecules such as nucleic acids or proteins on the basis of size, electric charge, and other physical properties.It can be performed within one dimension, two dimensions, or in a capillary. The sample is mixed into a buffer, and run on gels. Electrophoresis separations are usually carried out on gels made of agarose or polyacrylamide. These gels are chemically inert, so they will interfere little with the molecules. When electrophoresis is complete, the gel is stained to make the proteins visible. Common dyes used are Coommasie blue or silver staining. This method of protein separation and identification is useful because very little protein is needed to determine a difference in a protein. Distinct spots can be found with as little as 0.1 mg of protein when stained with Coommasie blue and even less (0.02 mg), is need when silver staining. Other methods of staining are Fluorescent dyes, and zinc or copper staining. Some drawbacks to this technique are that it is time-consuming and the purity of your protein sample will affect your results.
- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) - this method separates based on the mass of the molecule. Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins. The proteins are dissolved in SDS and then electrophorised. The smallest molecules move through the fastest, while larger molecules take longer and result in bands closer to the top of the gel.
- Isoelectric Focusing(IEF) - this method separates based on the isoelectric point(pI) of the protein. The isoelectric point is when the protein carries no net charge. The protein sample is applied to an immobilized pH gradient(IPG) strip. IPG strips come available over different lengths and pH ranges. As the sample is electrophorised, the proteins will migrate toward either the anode or cathode, depending on charge. The proteins will stop when they reach their respective pI, which results in a band. The band corresponds to a protein.
2D Electrophoresis is a technique that blends the two methods listed in One-Dimensional analyses. It is a very efficient separation technique for proteins. The protein sample is first run on an IPG strip, which after reaching completion is placed in either a horizontal or vertical SDS gel. This technique results in gels that contain spots. Each spot on the gel corresponds to a different protein. Then the gels are stained similarly as to 1D analysis. 2D Electrophoresis is widely used, and certain methods of it can be coupled with mass spectrometry in order to identify proteins.
Capillary Electrophoresis is very similar to 1D or 2D Electrophoresis, except it is performed within the small space of a capillary tube. This is advantageous in many ways. The heating that takes place due to high voltage loads on slab gels can have a negative effect on the separation of the proteins and the use of capillaries lessens said heat build-up. Also, because the gel will not need to be handled, it can allow one to use liquid polymers for separation, and can be replaced between runs. Automation is also much more possible with this technique, lessening the time, and making reproducible results.