Traditionally protein purification methods have often resulted in the separation of protein complexes for the sake of "purity" or have inadvertently destroyed complexes as a result of the chemical or physical basis of the separation procedure.
Sometimes the complex can be reconstituted by combining individually purified proteins, but in many cases activity cannot be recovered. In addition it is often difficult to identify the binding partners once they are physically separated.
Precipitation of a protein in an extract may be achieved by adding salts, organic solvents or organic polymers or by varying the pH or temperature of the solution.
Electrophoresis in free solution or in macro porous gels such as 1-2% agarose separates proteins mainly according to their net electric charge. Electrophoresis in gels such as poly-acrylamide separates mainly according to the molecular size of the proteins.
Separation by chromatography depends on the differential partition of proteins between a stationary phase and a mobile phase. Normally, the stationary phase is packed into a vertical column and the buffer is pumped through this column. An alternative is to stir the protein solution with the adsorbent. Pour the slurry onto the appropriate filter and make the washings and desorption on the filter.
Column chromatography has proved to be an extremely efficient technique for the separation of proteins in biological extracts.
In a typical fluidized bed there is a total mixing of particles and sample in the reactor leading to incomplete adsorption of the target molecules unless the feed stock is recycled. Expanded bed adsorption is a special case of fluidized bed adsorption and is primarily applied in a pilot or production scale environment.
In membranes, most pores allow convective flow, and the mass transport resistance is therefore minimized to film diffusion at the membrane matrix surface. The result is a more efficient adsorption-desorption cycle of target solutes, allowing considerably higher flow rates and thus considerably shorter separation times.
The simplest of all protein purification tags the HIS (histidine) tag is normally composed of six histidine residues. The DNA encoding these residues is cloned into the target gene such that the produced protein contains at some point in its polypeptide sequence six consecutive histidine residues.
Cloning is often performed such that the tag is located at either the extreme amino or extreme carboxy terminal end of the protein, where it is less likely to impair protein function.
The tag may, however also be placed in the middle of a protein if a central region is already known to be non essential for function.
A GST ( glutatione S - transferase) tag appends glutathione S-transferase to the protein of interest. Typical GST tags are on the order of 220 amino acids and can be used for protein purification by binding of the tagged protein to a solid support containing immobilized glutathione. Antibodies are available for immuno-histochemical analysis of proteins containing GST-tags.
Each and every preparation should be continually recorded in a purification table. In combination with results from gel electrophoresis, for example this will serve as a guide for judging the re productivity and outcome of each preparation.
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