Bio-Rad Profinity Epoxide Resin - Manual (Page 5)

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Section 2
General Considerations for Ligand Coupling
Removal of fines
Fine particles in resin may clog the column screen or filter membrane and increase
the column backpressure. Before Profinity epoxide resin is bottled, fines in the
medium have already been removed, so removal of fines is not necessary for most
applications. If necessary for a particular application, the very small amount of
remaining fines may be removed by decanting. Weigh out required amount of dry
resin in a hood (1 g of dry resin gives 5.5–8.0 ml of settled resin bed). Slurry resin
in 3 column volumes (CV) distilled water or buffer by agitation or mixing with a
paddle. Do not use magnetic stirrers, since magnetic stirbars will grind the resin
and more fines will be generated. Let resin settle for 25–40 min, and then carefully
decant the supernatant. The decanting process may be repeated a couple of
times if needed.
General protocols
Profinity epoxide resin can be used for coupling of a variety of ligands, such as
protein A, StrepTactin, subtilisin, and immunoglobulins. Since Profinity epoxide is
based on the UNOsphere platform, it has large pores. The medium’s open pore
structure is particularly useful for coupling large ligands and for purifying large tar-
gets. The general ligand coupling protocol is as follows:
1. Weigh out appropriate amount of dry resin (1 g dry resin swells to 5.5–8.0 ml
of settled resin in distilled water or buffers). Swell and wash resin with distilled
water or buffer. Do not use buffers containing Tris, glycine, or thiols since
these nucleophiles will compete with ligands for the resin’s active groups.
Removal of fines is not necessary for most applications. See Removal of fines
above.
2. Add protein ligand (5–20 mg per ml settled resin) to coupling solution. A
buffer: resin ratio of 1:1 to 2:1 is suitable for coupling. Salts such as
ammonium or potassium sulfate may be added to facilitate coupling.
3. Rotate the stoppered vessel containing the ligand and resin at ambient
temperature overnight (avoid using magnetic stirbars for mixing since the
resin’s physical properties will be damaged). Wash away unreacted ligand
using an ample amount of coupling buffer.
4. Collect ligand-coupled resin on a frit. Deactivate or block remaining active
groups by mixing resin cake with 1 M ethanolamine, pH 8–9 for at least
4 hours.
5. Wash the product thoroughly with coupling buffer. Additional wash cycles
alternating acidic and basic buffers are recommended. Each cycle should
consist of a wash of a pH 4.0 buffer (100 mM acetate, 500 mM NaCl)
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