Biomedical Engineering Reference
In-Depth Information
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Is the macromolecule of interest incorporated into organelles,
e.g., inclusion bodies, membranes, which afford (partial) de-
naturation?
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Are properties of the molecule of interest inhibited by compo-
nentsofthebuffers(e.g.,inhibitionofhemproteinsbysodium
azide or enzyme activity by SDS)?
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Is the monitoring of the purification process disturbed by buffer
components?
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Are properties of the molecule of interest regulated by divalent
cations and do chelators as EDTA therefore affect them?
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Is it necessary to add a stabilizator, such as, for example, sucrose
or glycerol?
The following of protocols for purification of similar macro-
molecules is very often successful; nevertheless, it is necessary to
optimize the procedure because it is possible that the given pro-
tocol will miss some essentials, or that the wanted macromolecule
will differ in important properties.
Before starting the purification of a more or less known macro-
molecule, it is helpful to collect information by the following:
Analytical methods
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Determination of molar mass by SDS-PAGE
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Determination of isoelectric point
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Identification of posttranslational modifications
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Determination of enzymatic parameters such as substrate
specificity, inhibitors, activators, pH optimum
Investigation of buffer compatibility
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Inhibition by buffer components
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Selection of stabilizators
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Renaturation after application of urea, guanidinium hydrochlo-
ride, or alcohols
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Stability in the presence of detergents
Test of possible chromatographic procedures
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Binding to specific ligands
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Binding to cation or anion exchanger at distinct pH and ionic
strength or to hydrophobic supports (binding may be as useful
as no binding if impurities show the opposite behavior)
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Significant differences in molecular size if gel filtration is in-
tended
As an example for combination of different methods in pro-
tein purification Fig. 3.2 gives the flow chart for the isolation of
membrane protein complex.
The classification of chromatography as gel permeation chro-
matography (GPC; size-exclusion chromatography, SEC; gel filtra-
tion, GF), ion exchange chromatography (IEC), hydrophobic inter-
action chromatography (HIC), and affinity chromatography (AC) is
