Electrophoresis is one of the widely used techniques in molecular biochemistry, microbiology, biomedical research.
It is a type of protein separation method which relies on protein sizes to segregate the mixture.
It is one of the highly effective techniques of analysis and sole method for separation of proteins for western blot, RNA studies, etc.
But, on the negative side it also time-consuming, expensive and technical skilled procedure due to which is less preferred in health care.
It is both qualitative and quantitative analysis technique.
Electrophoresis is similar to other separation techniques like chromatography, but it differs regarding the types of samples analyzed, the method used for separation, the principle used, etc.
Definition and Electrophoresis Principle
The term Electrophoresis means Electro=electric field + Phoresis=migration. So as the name indicates,
“electrophoresis is a method of separation wherein charged molecules migrate in differential speeds in an applied electric field.“
The charged molecules under the influence of electric field migrate towards oppositely charged electrodes.
Those molecules with a +ve charge move towards the cathode and -ve molecules move towards Anode. The migration is due to charge on the molecules and potential applied across the electrodes. The sample under test is placed at one end of the paper near one of the electrodes. When electricity is applied, the molecules start moving to respective electrodes.
But the movement is influenced by the molecular weight of the molecule. So when a mixture is placed on the electrophoresis paper or agarose gel, different bands are seen along the paper after the process.
This is due to the differential rate of movement by molecules based on their weight. Those molecules with higher molecular weight move slower. While those with small weight run1 faster. Also, the size of the molecule also influences the movement. The bigger size molecule experience more friction than smaller ones in motion. These molecules migrate at a different speed and to different lengths based on their charge, mass, and shape.
Types of electrophoresis & their techniques.
Electrophoresis can be broadly divided into two types as
- Slab electrophoresis
- Capillary electrophoresis.
The slab method is the classical method which is widely used for industrial scale. It is slow, time-consuming and bulky. Still, it is the sole method available for separation of proteins like enzymes, hormones, antibodies and nucleotides like DNA and RNA.
This slab electrophoresis is further divided into three types based on the principle used for separation.
a. Zone electrophoresis
Zone electrophoresis: Here the charged particles are separated into different zones or bands. This is of two types as
- Paper electrophoresis.
- Gel Electrophoresis.
Paper electrophoresis is a technique which employs a Whatman filter paper No.1 which is moistened by a buffer and then connected at two ends to two oppositely charged electrodes.
Then the sample is applied to one end and let for separation of components under electric gradients. After separation, the paper is dried and stained to get colored bands.
These colored bands are recognized for the nature of sample by comparing with the standard. For a sample of serum, five bands of proteins can be separated by paper electrophoresis.
Gel electrophoresis is a similar technique wherein instead of paper, a gel made of agarose or SDS (sodium dodecyl sulfate).
The separation is more efficient than paper type as the rate of the running of molecules is slow and area of separation is larger by thickness.
The sample is applied and subjected to an electric field which can lead to separation of molecules. These molecules form bands and can be recognized by staining and comparing with standard sample bands.
The method is more effective than paper and for instance from a serum sample, 15 proteins bands can be isolated.
Isoelectrofocusing: Here the isoelectric pH is set at different foci and hence the molecules are immobilized to their isoelectric point. They don’t move towards electrodes but stay at a specific isoelectric pH. This is even more efficient to separate proteins, and from serum, 40 bands of protein can be formed.
Immunoelectrophoresis: This is the method with the combination of principles of both electrophoresis with immune reactions. First, the proteins are separated on to the electrophoresis paper. Then the antibodies are allowed to diffuse through the paper and react with separated protein molecules in bands.
Also, read other immunoassay reactions for a better idea
Both of the methods are very specific and highly sensitive and widely used in microbiology.
As the name indicates, here the process of separation takes place inside a capillary tube.
The capillary electrophoresis is an advanced method of electrophoresis. This was developed with an intent to minimize the time taken for separation and analysis in slab electrophoresis.
This capillary electrophoresis requires a small sample in the range if 0.1 to 10 ηl while slab method requires in μl range. Also, this method yields high speed and high-resolution separations. Besides, the separated components which exit from one end of the capillary, are immediately analyzed by detectors fixed at the end of tubes.
The instrumentation is as below.
Applications of electrophoresis:
1. To separate complex molecules: Many complex biological molecules like vitamins B12. Antibiotics, proteins can be separated efficiently by electrophoresis. This is possible due to charge difference among the mixtures.
2. For analysis of nucleic acid molecules like RNA and DNA studies. These long chain molecules can be analyzed only after separation after electrophoresis. This helps to determine the size or breaks in the DNA or RNA molecule.
For more check out electrophoresis in practice for more methods and applications.