Affinity Chromatography - Department of Molecular and Cellular ...

1. Equilibrate the column with 5 column volumes of binding buffer.
2. Apply the sample.
3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by
UV absorption at A 280 nm ).
4. Elute with 5–10 column volumes of elution buffer. Collect fractions in neutralization buffer if low pH elution is
used*. The purified fractions can be buffer exchanged using desalting columns (see page 134).
*Since elution conditions are quite harsh, collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl,
pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral.
Since Benzamidine Sepharose 4 Fast Flow (high sub) has some ionic binding characteristics,
the use of 0.5 M NaCl and pH elution between 7.4–8.0 is recommended. If lower salt
concentrations are used, include a high salt wash step after sample application and before
elution.
The elution buffer used for competitive elution has a high absorbance at 280 nm. The eluted
protein must be detected by other methods, such as an activity assay, total protein or
SDS-PAGE analysis. The advantage with competitive elution is that the pH is kept constant
throughout the purification.
Cleaning
Wash with 3–5 column volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 followed with
3–5 column volumes of 0.1 M sodium acetate, 0.5 M NaCl, pH 4.5 and re-equilibrate
immediately with 3–5 column volumes of binding buffer.
Remove severe contamination by washing with non-ionic detergent such as 0.1%
Triton X-100 at +37 °C for 1 minute.
Media characteristics
Ligand density Composition pH stability* Mean particle
size
Benzamidine > 12 µmoles Amide coupling of ligand Short term 1–9 90 µm
Sepharose 4 p-aminobenzamidine/ml via a 14 atom spacer to Long term 2–8
Fast Flow highly cross-linked 4%
(high sub)
agarose
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects
on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place
and sanitization procedures.
Chemical stability
All commonly used aqueous buffers.
Storage
Wash media and columns with 20% ethanol in 0.05 M sodium acetate, pH 4.0 (use
approximately 5 column volumes for packed media) and store at +4 to +8 °C.
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