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Dye Ligand Chromatography 67
Fig. 1. Dye ligand chromatogram.
4. Notes
1. Prior to initiation of the resin screening, the protein of interest may be screened
for compatibility with planned binding and elution conditions. Understanding the
protein’s stability can be critically important to interpreting chromatographic data
during purification optimization. Dialysis of the protein against a range of buffers
followed by activity assays of each condition will define the borders of the
optimization space of the chromatography. Basic screening conditions for protein
activity include the following:
a. pH: 50 mM buffer + 100 mM NaCl, where the buffers are sodium acetate (pH
4 and 5), MES (pH 6) and HEPES (pH 7 and 8).
b. Salt/Detergent/Chaotrope/Solvent: Begin with 50 mM buffer + 100 mM NaCl,
where the buffer is chosen to give good protein activity. Add 0.01% Tween,
0.02% Triton X-100, 20% glycerol, 30% ethylene glycol, 1 M NaCl, 1 M
guanidine or 1 M urea to separate aliquots of the basic buffer. Verify protein
activity after exposure to each buffer. Use this information in selecting the wash
and elution conditions to be tested during batch screening.