Electrophoretic methods

Electrophoresis is an analytical method in which a controlled electric current is used in order to separate biomolecules according to their size ratio to electric charge, using a gelatinous matrix as the base. When a mixture of ionized and net charge molecules are positioned in an electric field, they experience a force of attraction towards the pole that has opposite charge, allowing the positively charged molecules to pass to the cathode (negative pole) positively charged will move towards the anode (positive pole).

The support on which the displacement of the species takes place is a polyacrylamide gel, this type of analysis is characterized because:

1. Migration is proportional to the net charge, size and shape of the protein.

2. Gels are chemically inert, transparent and stable over a wide range of pH, temperature and ionic strength.

3. Polyacrylamide gels (PAGE) are formed by the polymerization of acrylamide by the action of a cross-linking agent, bis-acrylamide, in the presence of an initiator (TEMED (N, N, N, N’-tetramethylnediamine) and as a catalyst. the persulfate ion (S2O8-), which is added in the form of ammonium persulphate.

Depending on the state of the proteins (native or denaturing) along the electrophoretic process, these are classified as native or denaturing electrophoresis:

In a denaturing electrophoresis: it is the one that subjects the proteins to migration ensuring complete denaturation (loss of the three-dimensional structure). In this situation the migration is proportional to the load and the size of the molecule but not to its shape.

In a native electrophoresis: the proteins are subjected to migration without denaturation. In this situation, proteins migrate according to their load, their size and their shape. In addition, the interactions between subunits and between proteins are maintained in certain cases, separating the complexes.


The use of electrophoresis by IEF-PAGE is limited to molecules that can be positively or negatively charged. Proteins, enzymes and peptides are amphoteric molecules. The net charge of a protein is the sum of all the positive and negative charges of the amino acid side chain, but the configuration of the protein also plays a role. For proteins such as glyco- or nucleoproteins, the net charge is also influenced by sugar or by the functional group of nucleic acid. The degradation of phosphorylation also has an influence on the net charge.

Isoelectric focusing is one of the best procedures for the isolation of proteins

The analysis by electrophoresis with SDS, is a type of natural electrophoresis in which the samples are denatured by heat in the presence of denaturing agents (beta-mercaptoethanol, which destroys disulfide bridges, SDS denatures and coats the protein), and they separate as isolated polypeptide chains. SDS is a denaturing detergent that binds denatured polypeptide chains with a ratio of 1.4g of SDS per gram of protein, one SDS molecule binding for every two amino acids in the chain. This massive union of SDS molecules blocks the charge of the molecule itself

protein and gives the complex a negative net charge proportional to its mass, causing all proteins complexed with SDS to travel to the anode.

DNA analysis

Electrophoresis is a way to analyze DNA, or deoxyribonucleic acid, which is the code that contains all inheritance characteristics. DNA is organized into sequences, for example, one sequence represents the color of the eyes and another sequence represents the color of the skin. By means of electrophoresis, specific DNA sequences can be analyzed, isolated and cloned. The analyzed DNA can be used in forensic investigations and paternity tests.

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