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Dye Ligand Chromatography 63
Development of a dye affinity chromatography step requires optimization
of binding, washing and elution conditions. This chapter describes both batch
chromatography (for scouting binding and elution conditions) and column
chromatography for purification optimization. The most efficient means of
establishing the binding conditions for a dye affinity purification is to use
batch chromatography. Use of this screening method can save substantial
time and expense by focusing the chromatographer’s efforts on the stationary
phase chemistries and the mobile phase conditions most likely to succeed (see
Note 1).
Having selected the appropriate dye affinity support and established the
possibility of quantitatively recovering protein activity, the chromatographer
can move onto column chromatography. This chapter describes a dye affinity
technique successfully used to purify a recombinant protein. Provided that the
reader takes the time to carry out the batch development successfully, the
adaptation of the column chromatographic technique should follow quickly.
2. Materials
2.1. Batch Binding
During buffer preparation, adjust pH to specified values using concentrated
hydrochloric acid or concentrated sodium hydroxide as appropriate.
1. Batch binding/wash buffers:
a. 20 mM sodium acetate, 50 mM NaCl, pH 4.
b. 20 mM sodium acetate, 50 mM NaCl, pH 5.
c. 20 mM MES, 50 mM NaCl, pH 6.
d. 20 mM HEPES, 50 mM NaCl, pH 7.
e. 20 mM HEPES, 50 mM NaCl, pH 8.
2. Batch elution buffers:
a. 20 mM sodium acetate, 1 M NaCl, pH 4.
b. 20 mM sodium acetate, 1 M NaCl, pH 5.
c. 20 mM MES, 1 M NaCl, pH 6.
d. 20 mM HEPES, 1 M NaCl, pH 7.
e. 20 mM HEPES, 1 M NaCl, pH 8.
3. Slide-A-Lyzer Dialysis Cassettes (Pierce, http://www.piercenet.com). Choose the
largest molecular weight cutoff that will not pass the protein of interest. The 3–12
ml size will allow one cassette per batch binding condition. A ratio of at least 100
to 1 should be maintained for the dialysis (1 l of dialysis buffer for each 10 ml of
sample to be dialyzed).
4. Dye ligand resins to be screened (see Subheading 1 for vendors).