Affinity Chromatography - Department of Molecular and Cellular ...

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Product Spacer arm Coupling conditions Maximum Comments
operating flow
HiTrap NHS-activated HP 10-atom pH 6.5–9, 15–30 min., 4 ml/min Pre-activated medium
+4 °C - room temp. (1 ml column) for coupling via primary
20 ml/min amine group of a ligand.
(5 ml column) Prepacked 1 ml and
5 ml columns.
NHS-activated 10-atom pH 6–9, 2–16 hours, 300 cm/h* Supplied as a suspension
Sepharose 4 Fast Flow +4 °C - room temp. ready for column packing.
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from
measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
Figure 59 shows that over 30 mg IgG can be coupled to a 1 ml HiTrap NHS-activated HP
column. The coupling process takes less than 15 minutes. The affinity medium is then ready
to use for antigen purification.
Protein coupled (mg)
40
30
20
10
20 40 60 80 100
Protein added (mg/1 ml column)
Fig. 59. Ligand coupling to HiTrap NHS-activated HP.
Preparation of HiTrap NHS-activated HP
The protocol below describes the preparation of a prepacked HiTrap NHS-activated HP
column and is generally applicable to NHS-activated Sepharose media. A general column
packing procedure is described in Appendix 3.
The activated matrix is supplied in 100% isopropanol to preserve the stability before
coupling. Do not replace the isopropanol until it is time to couple the ligand.
Buffer preparation
Acidification solution: 1 mM HCl (kept on ice)
Coupling buffer: 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3
Blocking buffer: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3
Wash buffer: 0.1 M acetate, 0.5 M NaCl, pH 4.0
Coupling within pH range 6.5–9, maximum yield is achieved at around pH 8.
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