Bio-Rad Profinity Epoxide Resin - Manual (Page 6)

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followed by a wash of a pH 8.0 buffer (100 mM phosphate, 500 mM NaCl).
The product can now be used for intended applications or stored in the
presence of a bacteriostat at 4–8°C for future use.
General coupling efficiency considerations
Amount of Ligand Used and Monitoring Amount Coupled
While the amount of ligand coupled is, to a certain extent, proportional to the
amount of ligand added to the coupling solution, the efficiency of ligand coupled
(varying with the ligand and conditions of coupling) will generally taper off at a
certain ligand concentration. In most cases, 5–20 mg of ligand per ml settled resin
is a good starting point to study optimal ligand concentration in a coupling
solution.
Soluble (unbound) ligand remaining in the coupling and wash buffers may be
monitored by measuring OD 280 or by using Bio-Rad protein assay kit II (catalog
#500-0002) or DC™ protein assay kit II (catalog #500-0112).
Coupling Buffers and pH
In order to maintain pH control, a minimum buffer strength of 10 mM is
recommended. Suitable buffers include carbonate, borate, and phosphate. Do not
use buffers such as Tris or glycine. They contain primary amino groups that will
couple to the resin, as will any other compounds containing nucleophiles.
Profinity epoxide couples ligands best at a pH range of 9–13. Often the choice of
coupling pH is limited by the stability of ligands in basic buffers. Profinity epoxide
resin’s epoxy groups react faster with ligands at a higher pH; however, competing
hydrolysis reactions of the resin’s epoxy groups also occur more frequently at
higher pH. Coupling of ligands through their hydroxyl groups requires a pH
around 13.
Coupling Temperature and Time
Coupling at 20°C is recommended for most applications. Carrying out the
coupling reaction at a higher temperature, up to 40°C, is normally faster if the
ligand is stable at the selected temperature. A water bath should be used to raise
the reaction temperature to the desired level.
Coupling ligands at ~20°C overnight is recommended for most applications. A
shorter coupling time is needed if both the pH of the coupling buffer and the
reaction temperature are high. A prolonged coupling reaction sometimes leads to
degradation of ligands and should be avoided.
Deactivation (Blocking) of Remaining Active Groups and Washing of
Ligand-Coupled Resin
Active groups remaining on the resin after coupling need to be deactivated or
blocked to avoid undesirable reaction with proteins of interest during affinity
chromatography. They can be deactivated or blocked by mixing the resin cake
with 1 M ethanolamine, pH 8–9 for at least 4 hr.
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