Affinity Chromatography - Department of Molecular and Cellular ...

Appendix 1
Sample preparation
Samples for chromatographic purification should be clear and free from particulate matter.
Simple steps to clarify a sample before beginning purification will avoid clogging the column,
may reduce the need for stringent washing procedures and can extend the life of the
chromatographic medium.
Sample extraction procedures and the selection of buffers, additives and detergents are
determined largely by the source of the material, the stability of the target molecule, the
chromatographic techniques that will be employed and the intended use of the product.
These subjects are dealt with in general terms in the Protein Purification Handbook and
more specifically according to target molecule in the Recombinant Protein Handbook,
Protein Amplification and Simple Purification and Antibody Purification Handbook,
available from Amersham Pharmacia Biotech.
Sample stability
In the majority of cases, biological activity needs to be retained after purification. Retaining
the activity of the target molecule is also an advantage when following the progress of the
purification, since detection of the target molecule often relies on its biological activity.
Denaturation of sample components often leads to precipitation or enhanced non-specific
adsorption, both of which will impair column function. Hence there are many advantages to
checking the stability limits of the sample and working within these limits during purification.
Proteins generally contain a high degree of tertiary structure, kept together by van der Waals'
forces, ionic and hydrophobic interactions and hydrogen bonding. Any conditions capable
of destabilizing these forces may cause denaturation and/or precipitation. By contrast,
peptides contain a low degree of tertiary structure. Their native state is dominated by
secondary structures, stabilized mainly by hydrogen bonding. For this reason, peptides
tolerate a much wider range of conditions than proteins. This basic difference in native
structures is also reflected in that proteins are not easily renatured, while peptides often
renature spontaneously.
It is advisable to perform stability tests before beginning to develop a purification protocol.
The list below may be used as a basis for such testing:
• Test pH stability in steps of one pH unit between pH 2 and pH 9.
• Test salt stability with 0–2 M NaCl and 0–2 M (NH 4 ) 2 SO 4 in steps of 0.5 M.
• Test the stability towards acetonitrile and methanol in 10% steps between 0 and 50%.
• Test the temperature stability in +10 °C steps from +4 to +40 °C.
• Test the stability and occurrence of proteolytic activity by leaving an aliquot of the sample
at room temperature overnight. Centrifuge each sample and measure activity and UV
absorbance at 280 nm in the supernatant.
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