Bio-Rad Media Sampler Pack - Manual (Page 13)

Elution
Elute target molecules with a step or a linear gradient. The salt concentration at
which the desired product elutes is predetermined with a linear gradient at small
scale. With this knowledge, the pH and salt concentration are adjusted to
eliminate the maximum amount of contamination before starting elution of the
target.
Regeneration
After each run, the packed bed should be washed with 2–4 CV of a high salt
buffer (0.5–2.0 M) to remove remaining bound material. It is important to do this
before acid treatment to avoid precipitation of protein on the media. If the column
no longer yields reproducible results, the medium may require more thorough
cleaning and sanitization.
Cleaning-in-Place (CIP)
Acceptable CIP agents include 1% acetic acid/1% phosphoric acid with 0.4 M
NaCl, up to 30% acetic acid, 1% Triton X-100, up to 70% ethanol or 30%
isopropyl alcohol, 8 M urea and 6 M guanidine-HCl. Any of these agents can be
combined in an appropriate cleaning protocol. As a general guide, we recommend
the following:
1. Use high salt buffer for regeneration, as above.
2. For aggregated or precipitated proteins, or when dirty feedstock (e.g., crude
lysate) had been used, wash with 3–5 CV of 6 M guanidine hydrochloride or
8 M urea at 100 cm/hr.
3. For lipids or hydrophobically bound contaminants, wash with 0.1% Triton X-
100, or 20–70% ethanol or isopropyl alcohol, or 10–30% acetic acid. Use
3–5 CV at 100 cm/hr.
4. Remove additional contaminants with 0.4 M NaCl in 1% acetic acid/1%
phosphoric acid (3–5 CV at 100 cm/hr).
5. If the column is to be used again immediately, wash with 2 CV of deionized
water and 4–5 CV of starting buffer at 100 cm/hr. Check the conductivity and
pH of the effluent to verify that the column is equilibrated in the starting buffer
before loading the sample.
Sanitization and Storage
To sanitize and store between campaigns, wash the column with 3–5 CV of
1% acetic acid/1% phosphoric acid, pH 1.5. Store the column at 4–40°C. When
not in use, store the Macro-Prep ion exchange supports in either 1% acetic acid in
1% phosphoric acid (pH 1.5) or in 20% (v/v) ethanol solution or in 2% benzyl
alcohol. The ion exchange supports may also be autoclaved at 121°C, 2 bar, in a
neutral pH slurry, for up to 30 min and stored in one of the above solutions.
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