Affinity Chromatography - Department of Molecular and Cellular ...

Table 3 gives examples of some typical binding and elution conditions that have been used
with Protein A Sepharose.
Table 3.
Binding to free Protein A Sepharose Protein A Sepharose
Species Subclass protein A binding pH elution pH
Usually elutes by pH 3
Human IgG 1 + + 6.0–7.0 3.5–4.5
IgG 2 + + 6.0–7.0 3.5–4.5
IgG 3 – 8.0–9.0 < 7.0
IgG 4 + + 7.0–8.0 use step elution
Cow IgG 2 + + 2
Goat IgG 2 + 5.8
Guinea pig IgG 1 + + 4.8
IgG 2 + + 4.3
Mouse IgG 1 + 8.0–9.0 5.5–7.5
IgG 2a + 7.0–8.0 4.5–5.5
IgG 2b + 7 3.5–4.5
IgG 3 + 7 4.0–7.0
Rat IgG 1 + > 9.0 7.0–8.0
IgG 2a – > 9.0 < 8.0
IgG 2b – > 9.0 < 8.0
IgG 3 + 8.0–9.0 3–4 (using thiocyanate)
Binding strengths are tested with free protein A. They can be used as a guide to predict the
binding behaviour to a protein A affinity medium. However, when coupled to an affinity
matrix the interaction may be altered. For example, rat IgG 1 does not bind to protein A,
but does bind to Protein A Sepharose.
IgGs from most species and subclasses bind protein A near to physiological pH and ionic
strength. Avoid excessive washing if the interaction between the protein of interest and the
ligand is weak, since this may decrease the yield.
With some antibodies, such as mouse IgG 1 , it might be necessary to add sodium chloride
up to 3 M in the binding buffer to achieve efficient binding when using protein A, for
example 1.5 M glycine, 3 M NaCl, pH 8.9.
Alternative elution buffers include: 1 M acetic acid, pH 3.0 or 0.1 M glycine-HCl, pH 3.0
or 3 M potassium isothiocyanate.
Potassium isothiocyanate can severely affect structure and immunological activity.
Use a mild elution method when labile antibodies are isolated. Reverse the flow of the wash
buffer and elute with 0.1 M glycyltyrosine in 2 M NaCl, pH 7.0 at room temperature,
applied in pulses. (Note: glycyltyrosine absorbs strongly at wavelengths used for detecting
proteins). The specific elution is so mild that the purified IgG is unlikely to be denatured.
To increase capacity, connect several HiTrap Protein A HP or HiTrap rProtein A FF
columns (1 ml or 5 ml) in series or pack a larger column with Protein A Sepharose 4 Fast Flow
or rProtein A Sepharose 4 Fast Flow (see Appendix 3).
36