Electrophoresis is a basic method in the field of molecular biology for the analysis (separation, purification, preparation) of nucleic acids and proteins. The principle of electrophoresis consists in the migration of the molecules through a gel or another type of matrix of a porous nature, in which, by the action of an electric field, they will be separated according to their size or molecular weight, this is achieved thanks to the action of a power source for electrophoresis.
There are two types of electrophoresis: vertical type electrophoresis, where both DNA and protein molecules are analyzed, while horizontal electrophoresis generally works with DNA or RNA.
The electrophoresis power source power is designed for use in DNA and RNA separation, RFLP, DNA fragmentation, preparative Page electrophoresis (polyacrylamide gel electrophoresis) and protein separation.
The basic procedure of electrophoresis
The process includes a matrix formed by starch, acrylamide, paper or some other substance capable of supplying a homogeneous support. On this matrix, protein or nucleic acid samples are placed. The infiltration of the samples into the matrix can be direct or through pieces of filter paper saturated with the protein mixture. The number of samples analyzed in a single run can vary from 10 to 50 depending on the type of electrophoresis.
The migration and separation of nucleic acids and soluble proteins in the sample is carried out by passing an electric current through the matrix for a certain time. The separation and migration speed depend on several factors, such as, among others, the electrical charge of the amino acids that make up the protein, the type of matrix and its thickness, the ionic strength and the composition of the buffer solutions. which are used to make the matrix.
In this way, proteins with a positive electrical charge migrate towards the negative pole (cathode) and proteins with a negative electrical charge migrate towards the positive pole (anode). Unlike proteins, which can have a positive or negative charge, nucleic acids only have a negative charge, due to their phosphate backbone. Therefore, in an electrophoresis, the nucleic acids will migrate towards the positive pole; that is, the anode.
Finally, once the proteins have migrated, their position is determined, applying a specific stain for the protein under study, which is generally an enzyme, and the same is done for nucleic acid electrophoresis. Two aspects are key in carrying out electrophoresis: first, a physicochemical part, which comprises the regulatory solutions used in the preparation of the matrix and the electrolytic solutions responsible for ion exchange and charge flow through the system; second, an electrical part related to the supply of current and voltage necessary for the separation of proteins.
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