Affinity Chromatography - Department of Molecular and Cellular ...

Chapter 2
Affinity chromatography in practice
This chapter provides guidance and advice that is generally applicable to any affinity
purification. The first step towards a successful purification is to determine the availability
of a suitable ligand that interacts reversibly with the target molecule or group of molecules.
Ready to use affinity media, often supplied with complete separation protocols, already
exist for many applications. The contents section of this handbook lists the full range of
affinity media from Amersham Pharmacia Biotech according to the specific molecule or
group of molecules to be purified. Application- and product-specific information and advice
for these media are supplied in other sections of this handbook. Practical information
specific to the use of pre-activated matrices for the preparation of affinity medium is
covered in Chapter 5.
Purification steps
1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific
binding of the target molecule(s) to a complementary binding
substance (the ligand). Target substances bind specifically,
but reversibly, to the ligand and unbound material washes
through the column.
3. Target protein is recovered by changing conditions to
favour elution of the bound molecules. Elution is performed
specifically, using a competitive ligand, or non-specifically, by
changing the pH, ionic strength or polarity. Target protein is
collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.
equilibration
adsorption of
sample and
elution of
unbound material
wash
away
unbound
material
elute
bound
protein(s)
re-equilibration
Absorbance
begin sample
application
change to
elution buffer
>1
1-2 cv x cv
1-2 cv cv 1-2 cv
Column Volumes (cv)
Fig. 3. Typical affinity purification.
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