Affinity Chromatography - Department of Molecular and Cellular ...

Performing a separation
Column:
Recommended flow rate:
HiTrap IgM Purification HP
1 ml/min
Binding buffer: 20 mM sodium phosphate, 0.8 M (NH 4 ) 2 SO 4 , pH 7.5
Elution buffer: 20 mM sodium phosphate, pH 7.5
Wash buffer:
20 mM sodium phosphate, pH 7.5 with 30% isopropanol
The sample must have the same concentration of ammonium sulphate as the binding buffer.
Slowly add small amounts of solid ammonium sulphate to the sample of hybridoma cell
culture supernatant until the final concentration is 0.8 M. Stir slowly and continuously.
Pass the sample through a 0.45 µm filter immediately before applying it to the column.
To avoid precipitation of IgM, it is important to add the ammonium sulphate slowly.
Purification
1. Wash column sequentially with at least 5 column volumes of binding, elution and wash buffer.
2. Equilibrate column with 5 column volumes of binding buffer.
3. Apply the sample.
4. Wash out unbound sample with 15 column volumes of binding buffer or until no material appears in the
eluent (monitored at A 280 ).
5. Elute the IgM with 12 column volumes of elution buffer.
6. Wash the column with 7 column volumes of wash buffer.
7. Immediately re-equilibrate the column with 5 column volumes of binding buffer.
Some monoclonal IgMs might not bind to the column at 0.8 M ammonium sulphate.
Binding can be improved by increasing the ammonium sulphate concentration to 1.0 M.
An increased concentration of ammonium sulphate will cause more IgG to bind, which
might be a problem if serum has been added to the cell culture medium. If there is IgG
contamination of the purified IgM, the IgG can be removed by using HiTrap Protein G HP,
HiTrap Protein A HP or HiTrap rProtein A FF.
Potassium sulphate (0.5 M) can be used instead of ammonium sulphate. Most monoclonal
IgMs bind to the column in the presence of 0.5 M potassium sulphate and the purity of IgM
is comparable to the purity achieved with 0.8 M ammonium sulphate.
Some monoclonal IgMs may bind too tightly to the column for complete elution in binding
buffer. The remaining IgM will be eluted with wash buffer, but the high content of
isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see
page 134) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol
may elute the IgM and decrease the risk of precipitation.
To increase capacity, connect several HiTrap IgM Purification HP columns in series.
HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid
chromatography system, such as ÄKTAprime.
Reuse of HiTrap lgM Purification HP depends on the nature of the sample and should only
be performed with identical samples to prevent cross-contamination.
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