Enzyme Technology
Gel exclusion chromatography
There is now a
considerable choice of materials which can separate proteins on the basis of
their molecular size. The original cross-linked dextrans (Sephadex G- series,
Pharmacia Ltd.) and polyacrylamides (Bio-Gel P- series, BioRad Ltd.) are still,
quite rightly, widely used. Both types are available in a wide range of pore
sizes and particle size distributions. However, as the pore size increases, for
use with larger enzymes, these gels become progressively less rigid and
therefore less suitable for large scale use. Consequently alternative, but
generally more costly, rigid gel materials have been developed for the
fractionation of proteins of molecular weight greater than about 75,000. These
are the cross-linked derivatives of agarose (Sepharose CL and Superose) and
dextran (Sephacryl S) made by Pharmacia Ltd., the cross-linked polyacrylamide-agarose mixtures
(Ultrogel AcA) made by LKB Instruments Ltd. and
the ethylene glycol-methacrylate copolymers (Fractogel HW) made by Toyo Soda
Company (TSK). These are available in a range of forms capable of fractionating
enzymes, and other materials, with molecular weights up to 108 and at
high flow rates. Although these gels are described as 'rigid', it should be
appreciated that this is a relative term. The best gels are significantly
compressible so scale-up from laboratory sized columns cannot be achieved by
producing longer columns. Scale-up is achieved by increasing the diameter of
columns (up to about 1 m diameter) but retaining the small depth. Further
scale-up is done by connecting such sections in series to produce 'stacks'.
Extreme care must be taken in packing all gel columns so as to allow even, well
distributed, flow throughout the gel bed. For the same reason, the end pieces of
the columns must allow even distribution of material over the whole surface of
the column. The newer materials are supplied in a pre-swollen state which enable
their rapid and efficient packing using slight pressure.
Gel exclusion
chromatography invariably causes dilution of the enzyme which must then be
concentrated using one of the methods described earlier.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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