Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Prime cartridge with 1 ml methanol.
- Equilibrate with 2 ml of water.
- Apply sample, collect flow through.
- Wash 3X with 400 µl of water, collect flow through.
- 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).
- Prime with 3 ml of acetonitrile (ACN) followed by 1 ml of 50% ACN.
- Equilibrate with 3 ml water.
- 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).
- Wash twice with 1 ml water.
- 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).