Affinity Chromatography Handbook

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To improve recovery of total IgY or a specific IgY antibody, replace 0.5 M K 2SO 4 with
0.6–0.8 M Na 2SO 4. The sample should have the same concentration of Na 2SO 4 as the
binding buffer.
An increase in salt concentration will reduce the purity of the eluted IgY.
The purity of the eluted IgY may be improved by using gradient elution with, for example,
a linear gradient 0–100% elution buffer over 10 column volumes, followed by elution with
100% elution buffer for a few column volumes.
To increase capacity, connect several HiTrap IgY Purification HP columns in series.
A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid
chromatography system such as ÄKTAprime.
Reuse of HiTrap IgY Purification HP depends on the nature of the sample. To prevent
cross-contamination, columns should only be reused with identical samples.
Media characteristics
Ligand and density pH stability* Mean particle size
HiTrap IgY Purification HP 2-mercaptopyridine Long term 3–11 34 µm
3 mg/ml Short term 2–13
*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.
Storage
Wash the column with 5 column volumes 20% ethanol and store at +4 to +8 °C.
Recombinant fusion proteins
The purification of recombinant proteins can often be simplified by incorporating a tag of
known size into the protein. As well as providing a marker for expression and facilitating
detection of the recombinant protein, an important role for the tag is to enable a simple
purification by affinity chromatography. The two most commonly used tags are glutathione-
S-transferase (GST) and 6 x histidine residues (His) 6. Protein A fusion proteins have also been
produced to take advantage of the affinity between IgG and protein A for affinity purification.
GST fusion proteins
GST MicroSpin Purification Module, GSTrap FF, GSTPrep FF 16/10, Glutathione
Sepharose 4 Fast Flow, Glutathione Sepharose 4B
Glutathione S-transferase (GST) is one of the most common tags used to facilitate the
purification and detection of recombinant proteins and a range of products for simple, one
step purification of GST fusion proteins are available (see Purification options).
Purification and detection of GST-tagged proteins, together with information on how to
handle fusion proteins when they are expressed as inclusion bodies, are dealt with in depth
in the GST Gene Fusion System Handbook and The Recombinant Protein Handbook:
Protein Amplication and Simple Purification, available from Amersham Biosciences.