*2.4.1 Protein isolation and purification*

The conventional methodology for the isolation of proteins includes the techniques for the separation of biochemical, where the protein is isolated from the other proteins based on its special chemical and physical properties. This involves molecular mass i.e. size, shape, net charge and hydrophobicity. The isolation procedure starts with the mixture of a cell extract is recognize in the course of the enzyme activity. This mostly involves crushing of the tissue in an extraction buffer and the contents of the cell [proteins] become accessible. To avoid proteolytic degradation of the enzyme, add protease inhibitors i.e. phenylmethyl sulphonylfluoride (PMSF), in the mixture of the extraction buffer. The first step is a centrifugation where the enzyme is precipitated or otherwise ends up in the supernatant which is bound to the cell wall or the cell membrane, Soluble proteins are usually separated from one another by their solubility in high-salt solutions with supported variation. Add a salt, followed by centrifuge to remove the precipitated proteins that will be fruitful strategy for proteins to separate from each other. During saturation, ammonium sulfate is usually used to precipitate proteins, as most of the proteins in the solution of this salt. The quantity of ammonium sulfate must be mixed in order to avoid subset of the proteins in the extract which can be determined by enzyme activity assays on the various fractions. Likewise, other salts, trifluoroacetic acid, protamine sulfate, polyethylene glycol, apolar solvents etc. are often used for the proteins precipitation. The next step used for further purifying the enzyme is chromatography such as High-performance liquid chromatography (HPLC), Hydrophobic interaction chromatography (HIC), Ion exchange chromatography(IEC) Bio-affinity chromatography (BAC), fast protein liquid chromatography (FPLC) and Gel filtration or size exclusion chromatography (GFC). When a fraction is obtained is the only protein during this procedure then purification is completed to homogeneity, or any contaminants are remained below the level of observation [8, 10, 11].
