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Analytical Ultracentrifugation Membrane Confined Analytical Electrophoresis Complementary Analytical Techniques
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Some ionic species, such as protein molecules away from the isoionic point, may pose problems arising from both charge and from the presence of bound ions. In order to maintain a constant pH, a buffer is normally used at concentrations between 10 and 50 mM. In addition, to suppress the non-ideality due to the charge in the macromolecule, a supporting electrolyte is usually added at 0.1-0.2 M KCl or NaCl. However, the presence of those extra salts means that the solution can no longer be treated as a simple two-component system, for which most theoretical relationships have been derived.
Fortunately studies show that if the macromolecular solution is dialyzed against a large excess of the buffer/salt solution, it may be treated as a two-component system. The sample should be dialyzed to equilibrium against the buffer.
Methods for achieving dialysis equilibrium:
1) Exhaustive dialysis (72 hours) against three changes (24 hours each) of solvent at a 100:1 vol: vol ratio.
2) Centrifugal gel filtration (Analytical Biochem, (1979) 100:184-187) or using centrifugal concentrators.
For absorbance optics, a sample of the dialysate is required as reference and for dilutions. During sedimentation experiments, the reference (buffer) absorbance will be subtracted (by the software) from the sample absorbance. If the dialysis does not reach equilibrium, the inherent difference between the buffer in the sample and the buffer in the reference creates an optical imbalance, which will contribute to and affect the absorbance vs. radius profile in further calculations. Imbalances in salt concentration may also give rise to Schlieren effects. Dialysis to equilibrium minimizes these effects and maintains the integrity of the data.
For interference optics, dialysis equilibrium must be reached. So long as the solution does not absorb strongly at 670nm, then any solvent components can be used. Note however, that some materials will not dialyze properly. This is especially true for detergents, in which case the absorbance system is the only choice.
Suggestions:
- Prepare the initial buffer solution with 20mM buffering ions using 150-200 mm salt for ionic strength.
- Example: sodium phosphate, pH 7.0, 200 mM NaCl.
- Use Tris only if the sample will be monitored at an absorbance of 280 nm. Tris interferes with protein detection at 230 nm.
- If a reducing agent is required, beta-mercaptoethanol, which does not absorb at 28nm, is preferred over DTT (dithiothreitol).
