Samples for chromatographic purifcation should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purifcation 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 Purifcation Handbook and more specifcally according to target molecule in the Recombinant Protein Handbook, Protein Amplifcation and Simple Purifcation and Antibody Purifcation Handbook, available from Cytiva.
In the majority of cases, biological activity needs to be retained after purifcation. Retaining the activity of the target molecule is also an advantage when following the progress of the purifcation, since detection of the target molecule often relies on its biological activity. Denaturation of sample components often leads to precipitation or enhanced non-specifc 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 purifcation.
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 purifcation protocol. The list below may be used as a basis for such testing:
Centrifugation and fltration are standard laboratory techniques for sample clarifcation and are used routinely when handling small samples.
It is highly recommended to centrifuge and filter any sample immediately before chromatographic purifcation.
Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after centrifugation, use filter paper or a 5 µm filter as a frst step and one of the filters below as a second step filter.
Filtration removes particulate matter. Membrane filters that give the least amount of nonspecifc binding of proteins are composed of cellulose acetate or PVDF.
For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium.
Check the recovery of the target protein in a test run. Some proteins may adsorb non- speciﬁcally to ﬁlter surfaces.
Desalting columns are suitable for any sample volume and will rapidly remove low molecular weight contaminants in a single step at the same time as transferring the sample into the correct buffer conditions. Centrifugation and/or ﬁltration of the sample before desalting is still recommended. Detailed procedures for buffer exchange and desalting are given on page 133 (see page 133, Buffer exchange and desalting for affinity chromatography).
At laboratory scale, when samples are reasonably clean after ﬁltration or centrifugation, the buffer exchange and desalting step can be avoided. For afﬁnity chromatography or hydrophobic interaction chromatography, it may be sufﬁcient to adjust the pH of the sample and, if necessary, dilute to reduce the ionic strength of the solution.
Rapidly process small or large sample volumes. Use before and/or between puriﬁcation steps, if needed (remember that each extra step can reduce yield and desalting also dilutes the sample).
Remove salts from proteins with molecular weight Mr > 5 000.
Use 100 mM ammonium acetate or 100 mM ammonium hydrogen carbonate if volatile buffers are required.
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