5. Protein purification
There are many thousand kinds of proteins with different properties and functions in a cell. Proteins have been purified in active form on the basis of such characteristics as solubility, size, charge and specific binding affinity. Normally, protein mixtures are subjected to a series of separations, each based on a different property to yield a pure protein.
5.1 Separation based on solubility (Scopes, 1994)
The protein solubility in various solvents depended on the distribution of hydrophilic and hydrophobic residues on the surface of the protein molecules. The properties of solvent can be manipulated to change protein solubility by alters in pH, ionic strength, other solutes or polymers, addition of miscible …show more content…
Then, the supernatant is used for further purification steps. To know the location of the desired protein in cell, the differential centrifugation or density gradient centrifugation is a good choice for protein separation in the each organelle. In differential centrifugation, the particles are separated according to their sizes into fractions by stepwise increase of the centrifugal force. Density gradient centrifugation is a scheme to separate particles by their densities. Several materials can be used to make the gradients such as sucrose and cesium chloride (Ohlendieck, 2005).
5.2.2 Dialysis
This method is simple, but time consuming because the separation depends on diffusion. Conventional use of dialysis bags involves the discharge of unwanted low-molecular weight solute from the sample and replacement with buffer present in the dialysate. Dialysis tubing is available in a variety of sizes and does not allow molecules larger than 15-20 kDa to pass through the membrane. Moreover, dialysis is also used to concentrate protein solution. Water molecules are removed from the inside of the dialysis bag by using hydrophilic polymer such as polyethylene glycol.
5.2.3 Ultrafiltration (molecular
There are many thousand kinds of proteins with different properties and functions in a cell. Proteins have been purified in active form on the basis of such characteristics as solubility, size, charge and specific binding affinity. Normally, protein mixtures are subjected to a series of separations, each based on a different property to yield a pure protein.
5.1 Separation based on solubility (Scopes, 1994)
The protein solubility in various solvents depended on the distribution of hydrophilic and hydrophobic residues on the surface of the protein molecules. The properties of solvent can be manipulated to change protein solubility by alters in pH, ionic strength, other solutes or polymers, addition of miscible …show more content…
Then, the supernatant is used for further purification steps. To know the location of the desired protein in cell, the differential centrifugation or density gradient centrifugation is a good choice for protein separation in the each organelle. In differential centrifugation, the particles are separated according to their sizes into fractions by stepwise increase of the centrifugal force. Density gradient centrifugation is a scheme to separate particles by their densities. Several materials can be used to make the gradients such as sucrose and cesium chloride (Ohlendieck, 2005).
5.2.2 Dialysis
This method is simple, but time consuming because the separation depends on diffusion. Conventional use of dialysis bags involves the discharge of unwanted low-molecular weight solute from the sample and replacement with buffer present in the dialysate. Dialysis tubing is available in a variety of sizes and does not allow molecules larger than 15-20 kDa to pass through the membrane. Moreover, dialysis is also used to concentrate protein solution. Water molecules are removed from the inside of the dialysis bag by using hydrophilic polymer such as polyethylene glycol.
5.2.3 Ultrafiltration (molecular