Home Protocols Glycan SPE C18 and Graphitized Carbon Protocols (P0710)

Glycan SPE C18 and Graphitized Carbon Protocols (P0710)

To remove peptides, protein, and other contaminants present in a glycan sample, use a C18 cartridge such as the Supelclean™ ENVI-18 (bed wt. 100 mg, volume 1 ml).
  1. Prime cartridge with 1 ml methanol.
  2. Equilibrate with 2 ml of water.
  3. Apply sample, collect flow through.
  4. Wash 3X with 400 µl of water, collect flow through.
  5. Combine all fractions. 

To remove salts and other small molecules, use a graphitized carbon cartridge such as the ENVI-Carb™, (bed wt. 100 mg, volume 1 ml).

  1. Prime with 3 ml of acetonitrile (ACN) followed by 1 ml of 50% ACN.
  2. Equilibrate with 3 ml water.
  3. Apply glycan sample (directly after deglycosylation, or after C18 SPE). Glycans will be retained on the graphite resin (di- or trisaccharides may not bind, monosaccharides will not bind).
  4. Wash twice with 1 ml water.
  5. Elute with 300 µl of 40% ACN/60% 50mM Ammonium Formate pH 4.4 (low pH is needed to elute sialylated glycans).
    Note: To elute very large glycans higher acetonitrile concentrations might be necessary. Lyophilize, or dry in speedvac (to prevent sialic acid loss, do not overheat).