Protein Protocols Protein Protocols

822 Hounsell, Davies, and Smith
3. Methods
1. Prepare eluant A: 100 mM NaOH, 500 mM Sodium acetate.
2. Prepare eluant B: 100 mM NaOH.
3. Prepare the column by elution with 50% A/50% B for 30 min at a flow rate of 1 mL/min.
4. Equilibrate column is 5% eluant A/95% eluant B at a flow of 1 mL/min.
5. Connect the AMMS2 to eluant out line and autoregen unit containing 500 mL of 50 mM
reagent-grade H 2SO 4, and pump regenerant at a flow of 10 mL/min (see Note 1).
6. Inject 200 pmol of each oligosaccharide or sialylated oligosaccharide alditol (more if
required for NMR or LSIMS), and elute with the following gradient at a flow of 1 mL/min:
a. Time = 0 min A = 5%; B = 95%
b. Time = 15 min A = 5%; B = 95%
c. Time = 50 min A = 40%; B = 60%
d. Time = 55 min A = 40%; B = 60%
e. Time = 58 min A = 0%; B =100%
7. Equilibrate the column in 5%A/95%B prior to the next injection.
8. At the end of the analyses, regenerate the column in 100%, and flush at the pumps
with H 2O.
9. Desalt oligosaccharide-containing fractions by AMMS, and derivatize for LSIMS
and GC-MS.
4. Notes
1. A better desalting profile may be achieved with an AMMS membrane (rather than
AMMS2) if a flow rate of