Protein Purification Protein Purification Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps may exploit differences in (for example) protein size, physicochemical properties, binding affinity and biological activity. Protein Purification Methods Complex protein mixtures were fractionated mainly by adsorption and precipitation methods. These methods are still used today as preliminary steps for initial fractionation or for concentration of sample solutions.
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Preparative electrophoretic and chromatographic techniques developed during the 1950's and 1960's made possible rational purification protocols and laid the foundation for the situation. The following are the methods used for the protein purification. Precipitation 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 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 polyacrylamide separates mainly according to the molecular size of the proteins. Chromatography 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. Expanded bed adsorption 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. Membrane adsorption
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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.
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