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Dye Ligand Chromatography 69
6. Secondary screening: Frequently, secondary screening for elution conditions is
useful. Dye affinity resins may bind the protein of interest with high affinity
and prevent good product recovery using the initial elution conditions. In the
secondary screen, focus on the resins that have generated reasonable capacities for
the protein of interest during primary screening. Carry out binding on these resins
using the optimal conditions found in the initial binding study, then vary the elution
condition either by variation in pH or conductivity or through the addition of other
elution modulators: 0.01% Tween, 0.02% Triton X-100, 20% glycerol, 30% ethylene
glycol, 1 M guanidine and 1 M urea, provided that each of these is compatible
with protein activity/stability. The goal is to find an elution condition capable of
eluting the protein of interest quantitatively while preserving any relevant biological
characteristics (full activity, native structure, etc.). In some cases, extremely high
affinity between the ligand and the target protein may preclude the identification
of suitable elution conditions. Usually, a moderate affinity dye ligand can be found
which will release active protein. The chances of finding a moderate affinity dye
ligand are enhanced by screening a large number of resins initially.
References
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Protein and Enzyme Technology, MacMillan Press, London.
3. N.E. Labrou. (2003) Design and Selection of Ligands for Affinity Chromatography.
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4. R. M. Chicz and F. E. Regnier. (1990) High Performance Liquid Chromatography:
Effective Protein Purification by Various Chromatographic Modes. Methods
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Enzymol. 182, 24–38.
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