Proteins are the most important biomacromolecule present in each and every living cell. Proteins play a major role in the survival of all the cells. These proteins are present in our body in many forms such as hair, cartilage, tendons, ligaments, muscles and skin. Proteins are made up of polypeptide that is each protein is made up of linear chains of amino acids. These amino acids are linked together by a bond known as peptide bonds.
Basis of Protein Purification:
To know about the working mechanism of a protein, it is very much essential to know about its amino acid sequence and also their nature. Once the purification of a protein is completed, then it is very easy to determine the amino acid sequence. Proteins are purified using appropriate purifying techniques. Purified sample of protein contains only one type or required type of protein.
First proteins are resealed from the cell and then they are separated depending on their densities using a method known as differential centrifugation. After this separation proteins can be differentiated from one another on the basis of their size, charge, solubility and also binding capacity. Most popular and widely used method of protein purification is discussed below.
Chromatography is a technique used to separate the organic compounds based on their size, charge they posses, shape and also solubility. Chromatography usually consists of a mobile phase and a stationary phase. Stationary phase consists of either a paper or a Colum via which the mobile phase travels. Mobile phase consists of a solvent and the biomolecules to be separated from the mobile phase. Depending upon chemical nature of the biomolecule, they travel through the stationary phase at different rate or speed.
Ion -exchange Chromatography:
Ion-exchange chromatography relies on the surface charge present in the protein of interest for separation. It uses the charge-charge interaction between the protein present in the sample and the charges immobilized on the resin used as stationary phase.
Around two third of the dry matter of living things contains protein. Therefore efforts are made to purify a protein of interest from other proteins present in a living tissue or a cell. Knowledge about the chemistry of protein of interest like molecular weight, size, shape and also surface charge are required for the chromatographic separation.
Gel Filtration Chromatography:
Gel filtration chromatography uses the principle of gravity for the separation of proteins. In this technique proteins are separated based on their size. In gel filtration chromatography microscopic glass beads with small holes are used to form the column.
A solvent sample contain the desired protein is passed through the column. Molecules that are smaller than the holes present in the glass beads get suspended up in the beads. Therefore, only the smaller molecules move via the column comparatively slower than the larger molecules. That is based on the size of the biomolecule protein they are separated in this technique.
In affinity chromatography, antibody specific to the protein of interest or protein to be purified is attached to the glass beads, and are used as stationary phase. Then mixture of proteins is added to the column.
Protein to be purified will bind to the specific antibody and all other biomolecules passes through the column.
High - Pressure Liquid Chromatography:
In this technique mobile phase used is a liquid. Stationary phase is made up of powdered solid absorbents, which are packed into a thin metal column. The mobile phase consists of an eluting solvent, which is then forced into the column by using a high pressure pump. The mixture of biomolecules to be separated and analysed is injected into the column. A detector is used to monitor this process.
Dialysis is a simple and easy technique used in separating the biomolecule protein. This technique involves a semipermeable membrane such as cellulose. The biomolecules which are having a dimension more than that of cellulose pore size will remain inside the cellulose membrane. Whereas smaller molecules emerge out of the cellulose membrane. But this technique is not considered as an effective method for protein separation.
Salting out Technique:
This technique uses the basic principle such as in a definite concentration of salt, the ability to precipitate differs from one protein type to another. This technique is also used to concentrate the dilute solutions of proteins, which are separated using any other protein purification techniques.
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