Wheat germ lectin can be used for group speciﬁc afﬁnity puriﬁcation of glycoproteins and polysaccharides. This lectin binds N-acetylglucosamine residues and reacts strongly with the chitobiose core of N-linked oligosaccharides. It also has afﬁnity for N-acetylneuraminic acid. Wheat germ lectin is a dimeric, carbohydrate-free protein composed of two identical subunits, each with a molecular weight of approximately Mr 20 000.
*Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
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.
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.
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.
* 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.
Avoid exposure to conditions below pH 4.0 as this causes dissociation of the wheat germ lectin dimer.
Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.
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