Affinity Chromatography - Department of Molecular and Cellular ...

1. Pack the column (see Appendix 3) and wash with at least 5 column volumes of binding buffer.
2. Equilibrate the column with approximately 5 column volumes of binding buffer.
3. Wash with 2–3 column volumes of acetic acid followed by 5 column volumes of binding buffer.
4. Apply the sample.
5. Wash with 10 column volumes binding buffer.
6. Wash with 2 column volumes of wash buffer or until no material appears in the eluent (determined by UV
absorbance at A 280 nm ).
7. Elute with 2–5 column volumes of elution buffer.*
8. Immediately re-equilibrate the column with binding buffer until the eluent reaches pH 7.0 (the IgG may
denature if left at a lower pH).
*Since elution conditions are quite harsh, it is recommended to 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.
This method, while giving a concentrated eluate, can only be used if the fusion product is
stable under the acid conditions.
An alternative eluent is 0.1 M glycine-HCl, pH 3.0. Chaotropic agents may also be used
for elution.
Media characteristics
Ligand Composition pH stability* Particle size
IgG Sepharose 6 Fast Flow Human polyclonal IgG IgG coupled to Short term 3–10 90 µm
Sepharose Fast Flow Long term 3–10
by the cyanogen
bromide method.
*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
Avoid reducing agents such as 2-mercaptoethanol or DTT since they may disrupt disulphide
bonds within the IgG ligand.
Storage
Wash with 5 column volumes of 20% ethanol at neutral pH and store at +4 to +8 °C.
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