Protein adsorption final stage

Hydroxylapatite column chromatography relies on the selective adsorption of the protein onto the surface of calcium phosphate. The final stage of purification, on a high-performance DEAE column, is carried out just prior to crystallization of the protein. 

The most convenient and simple method for production is ultrafiltration. The method uses membrane tubes with pore sizes from about 6000 to 50000 A. Small molecules like salt ions as well as water pass through the pores of the membrane tube while large molecules like proteins remain inside the tube. The concentration of the buffer solution is the same before and after ultrafiltration. The leaking of desired proteins in permeates should be checked during the concentration stage. Regular maintenance is carried out by using a standard protein. The membrane tube is made of polyethylene, polypropylene etc., and the irreversible adsorption of desired proteins should be avoided. The materials for the membrane should be selected before use. The flow rate of ultrafiltration depends on the facility and the protein solution applied. The final protein concentration is up to about 100 g/L. 

A major area for new research concerns the structural and functional consequences of adsorption of proteins to surfaces (items 3-5 in Table IV). Measurement of conformational change is still in an early stage of development. Most methods for studying adsorbed protein conformation are restricted to comparison of spectral differences induced by adsorption, without knowledge of the actual type or amount of change these differences reflect. Better methodology, especially on quantitative aspects, is sorely needed in this area. The orientation of adsorbed proteins may prove to be readily explored with the monoclonal antibody method and therefore certainly deserves wider application. Finally, the behavior of enzymes and antibodies at interfaces is not... 

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