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66 Gallant et al.
10. Data interpretation:
a. Binding: The primary event to be looked for is binding. Provided reasonable
protein capacity is achieved, the protein can usually be eluted with one of
the strategies mentioned in Table 1. Look for samples in which little activity
remains in the binding supernatant.
b. Elution: In the initial screen described above, both pH and sodium chloride
are examined as possible eluents. Look for pH conditions and sodium chloride
conditions at which the protein is eluted and is found in the supernatant.
3.2. Dye Ligand Chromatography
For the protein purification by dye ligand chromatography described below,
the following conditions are used:
– Binding condition: Cell culture supernatant titrated to pH 5 with 10% acetic acid;
binding to GE Amersham Blue Sepharose 6 FF.
After binding, the pH is increased to 6.5 without eluting the protein, while
the sodium chloride concentration remains low. The wash condition and the
elution condition were the following:
a. Wash condition: 10 mM sodium phosphate, pH 6.5 (Gradient Buffer A).
b. Elution condition: A linear gradient between Gradient Buffer A and Gradient
Buffer B (10 mM sodium phosphate, 1 M NaCl, pH 6.5).
The method employed in the chromatography is as follows:
1. A loading of 0.78 mg target protein/ml of packed resin is used. Seventy milligrams
of the protein of interest is loaded on a 90-ml column (17 × 2.6 cm).
2. A flow rate of 13.3 ml/min is used. This flow rate is quite conservative and the
manufacturer would allow up to five times the flow rate based on this column’s
cross-sectional area. See individual manufacturer’s resin specifications.
3. Consult the manufacturer’s instruction to pack the column.
4. Sanitize the column by passing 3 column volumes of cleaning buffer (0.1 M
NaOH) at 13.3 ml/min.
5. Equilibrate the column at 13.3 ml/min with 3 column volumes of binding buffer
and check the eluent pH. Repeat until pH is correct.
6. Load the sample at 13.3 ml/min.
7. Wash with 10 column volumes of Gradient Buffer A or until detector baseline
(typically A 280 nm ) is reached.
8. Run a linear gradient at 13.3 ml/min from 0 to 100% B in 20 column volumes.
Collect fractions of 0.5 column volume.
9. Repeat sanitization and store in 20% ethanol or equivalent bacteriostatic solution.
10. Analyze fractions for activity.
11. Data analysis: In the example chromatogram (see Fig. 1), 85% of the activity
was recovered in main A 280 nm peak (factions 6–14).