Affinity Chromatography - Department of Molecular and Cellular ...

Purification options
Binding capacity/ml Maximum Comments
medium
operating flow
Agarose Wheat Germ Lectin No data available 75 cm/h* Supplied as a suspension
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.
Performing a separation
Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Elution buffer: 0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4
Agarose Wheat Germ Lectin can be used with detergents, such as 1% deoxycholate or
0.5% Triton X-100.
1. Pack the column (see Appendix 3) and wash with at least 10 column volumes of binding buffer to remove
preservative.
2. Equilibrate the column with 10 column volumes of binding buffer.
3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most
significant factor for maximum binding).
4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by
UV absorption at A 280 nm ).
5. Elute with 5 column volumes of elution buffer.
Use 0–0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 with a continuous
gradient or step elution to improve resolution of complex samples containing glycoproteins
with different affinities for the lectin.
Elute tightly bound substances with 20 mM acetate buffer, pH 4.5 or with an alternative
sugar, for example triacetylchitotriose.
Higher concentrations of eluting substances may be necessary and recovery may be improved
by pausing the flow for some minutes during elution.
Cleaning
Wash with 5–10 column volumes of 20 mM Tris-HCl, 1 M NaCl, pH 8.5 and re-equilibrate
immediately with binding buffer. Low concentrations of non-ionic detergents in the Tris-HCl
buffer can be used if necessary, for example 0.1% Nonidet P-40.
Media characteristics
Ligand density Composition pH stability* Mean particle
size
Agarose Wheat Germ Lectin 1–2 mg/ml Wheat Germ Lectin Short term 4–9 90 µm
coupled to Sepharose 4B Long term 4–9
by CNBr method
*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.
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