rGRFT-LecBeads [for Manα(1-2)Man]

< Preparation >
Buffers
Binding buffer : PBS pH7.5
Elution buffer : PBS pH7.5 containing 200 mM D-mannose [Product code: M0045] or methyl α-D-mannopyranoside [Product code : M0368] and 0.1% triton-X100 [Product code: P1775]

< Method of the lectin-catch > ※Centrifuge method

Equilibration

• Prepare the LecBeads in a new tube.
• Add the Binding buffer into the tube to equilibrate the gel.
• Centrifuge the tube at 800 ×g for 3 min and discard the supernatant. (If necessary, repeat equilibration with the Binding buffer >10-fold volume of the LecBeads appropriately.)

Binding

• Concentrate or dilute the glycoprotein sample to moderate concentration (e.g., 1 mg/ml) with the Binding buffer [Input fraction], and apply the prepared sample to the tube in which the LecBeads was preliminary equilibrated.
• Agitate the tube at 4°C (※Generally, a dissociation constants (Kd value) of lectins are tend to be decreased at lower temperature) for a few hours (or overnight).
• Centrifuge the tube at 800 ×g for 3 min and remove the supernatant [Through fraction].
• Wash the LecBeads by 5 (to 10)-fold of the slurry volume with the Binding buffer several times.

Elution

• Elute the glycoprotein from the LecBeads by agitation with an appropriate volume of the Elution buffer (e.g. same volume of the LecBeads).
• Agitate the reaction tube at 4°C or R.T. for a few hours (or overnight).
• Centrifuge the tube at 800 ×g for 3 min and remove the supernatant [Elution fraction].
• Check the resulting fractions ([Input fraction] [Through fraction] [Elution fraction]) by protein assay and SDS-PAGE. (In some cases, the repetitive elution step with a small volume of the Elution buffer might improve the elution effect. If necessary, repeat elution steps.)
• The competitive oligosaccharide included in the obtained elution fractions can be removed by ultrafiltration or dialysis using a sufficient amount of buffer.

References

• Glyco-catch method: A lectin affinity technique for glycoproteomics
  • J. Hirabayashi, T. Hashidate, and K. Kasai, J. Biomol. Technol. 2002, 13, 205.
• Separation technologies for glycomics
  • J. Hirabayashi, K. Kasai, J. Chromatogr. B, 2002, 771, 67.