What are the principles of electrophoresis?

June 29, 2022by Kalstein

This technique is widely used in laboratories, especially in those of molecular biology, because it is used in important procedures such as: separation, analysis and purification of RNA, DNA, or proteins, nucleic acids, this process is performed because most biomolecules have an electrical charge where their magnitude depends on the pH of the medium in which they are found; because of this, the biomolecules move when subjected to an electric field to the charge pole opposite to that of the molecule.

Its operation is a simple process, separates the molecules according to their size and electrical charge, for this an electric current is used that impels to displace the molecules through a gel or another matrix., then the pores of the gel act as a sieve, allowing the smaller molecules to move faster than the larger molecules, to know the size of the molecules in a sample, they are compared with the established size standards that are separated into the same gel.

As it happens of molecule migration in electrophoresis.

In the case of proteins that can have a positive or negative charge, are usually neutral charge, for the migration of molecules pretreatment with detergents, such as sodium dodecyl sulfate (SDS), which confers negative charge; this homogenizes the proteins in the sample and will all migrate to the positive pole; will only be separated by size.

Now on the side of nucleic acids only have a negative charge, because of their phosphate skeleton, instead electrophoresis, causes nucleic acids to migrate towards the positive pole, called anode. This migration technique is what we call the principle of electrophoresis, where the displacement of the molecules through a gel or other type of porous matrix occurs proportionally, using the parameters of molecular weight or size; movement generated by the electric field.

Principles of electrophoresis.

This principle is based on the techniques used for the migration of molecules, this process is performed in a kind of box which has a charge with a positive end on one side and a negative charge on the other side, when placed inside a charged molecule, it moves following a specific pattern, that is, if the molecules have negative charge they will migrate to the positive charge, but on the contrary if it is positively charged as it will move to the negative side.ย 

When you analyze the proteins in a gel you take the whole protein to see how big it is, meeting the next principle that the shorter, the more they migrate into the gel, so that the small proteins will end up at the bottom of the gel, because they’ve gone further, and the larger ones will end up staying at the top. But in the case of DNA, we work with a very long molecule, so scientists opt to cut DNA with things like restriction enzymes, making DNA more manageable into smaller pieces, then depending on how big they are, they migrate more or less through the gel and end up higher or lower.

Elements needed for electrophoresis

To perform an electrophoresis requires a number of elements, such as: electrophoresis chamber, combs for wells, gel, transiluminator, power source, current buffer, molecular weight marker, charge buffer; it is important to note that the chambers of vertical electrophoresis the positive pole is located in the lower part of the chamber and in the horizontal, in one of the ends. In both types of cameras the positive pole is identified with red and the negative with black.

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