Affinity Chromatography - Department of Molecular and Cellular ...

Media characteristics
Ligand and density pH stability* Mean particle size
HiTrap IgM Purification HP 2-mercaptopyridine Long term 3–11 34 µm
2 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.
Avian IgY from egg yolk
HiTrap IgY Purification HP
Purification option
Binding capacity Maximum Comments
operating flow
HiTrap IgY Purification HP 100 mg pure IgY/column 20 ml/min Purification of IgY from egg yolk.
(1/4 egg yolk) Prepacked 5 ml column.
HiTrap IgY Purification HP columns are packed with a thiophilic adsorption medium
(2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the
protein and the ligand has been suggested to result from the combined electron donatingand
accepting-action of the ligand in a mixed mode hydrophobic-hydrophilic interaction.
Purification example
Figure 20 shows the purification of a-Hb IgY from 45 ml of egg yolk extract (corresponding
to one quarter of a yolk) and Figure 21 shows the SDS-PAGE analysis indicating a purity
level of over 70%.
A 280 nm
IgY
mS/cm
Sample: 45 ml of egg yolk extract (corresponding to 1/4 of
an egg yolk) containing a-Hb IgY, filtered through
a 0.45 µm filter
Column: HiTrap IgY Purification HP, 5 ml
Binding buffer: 20 mM sodium phosphate buffer,
0.5 M potassium sulphate, pH 7.5
Elution buffer: 20 mM sodium phosphate buffer, pH 7.5
Cleaning buffer: 20 mM sodium phosphate buffer,
pH 7.5, 30% isopropanol
Flow:
5 ml/min
2.0
1.0
Elution
buffer
Cleaning
buffer
80
60
40
20
0
0
0 50 100 150 ml
Fig. 20. Purification of avian IgY on HiTrap IgY Purification HP.
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