Protein Protocols Protein Protocols

870 Merry
4. Dialyze for a minimum of 48 h in a microdialysis apparatus fitted with a 6000–8000-
Dalton cutoff membrane.
5. Recover sample from dialysis membrane. Wash membrane with 0.1% TFA to ensure
recovery.
6. Transfer to a suitable tube for hydrazinolysis (see Note 9).
7. Lyophilize sample for 48 h.
8. For O-glycan analysis further drying is recommended (see Note 10).
9. Remove sample from lyophilizer immediately prior to addition of hydrazine.
3.1.3.2. MANUAL HYDRAZINOLYSIS PROCEDURE
Suitable for analysis of N- and O-linked glycans when expertise and equipment for
the procedure are available (34).
1. Remove tubes from drying immediately prior to hydrazine addition.
2. Flush tube with argon, taking care not to dislodge lyophilized protein.
3. Rinse dried syringe fitted with stainless steel needle with anhydrous hydrazine.
4. Take up fresh hydrazine and dispense onto sample. A 0.1-mL volume of hydrazine is
sufficient to dissolve up to 2 mg of glycoprotein. For larger amounts add more hydrazine.
5. Seal the tube.
6. Gently shake tube—the protein should dissolve.
7. Place in an incubator (do not use water bath).
8. For release of N-linked glycans incubate at 95°C for 5 h; for O-glycan release incubate for
60°C for 6 h.
9. Allow to cool and remove hydrazine by evaporation.
10. Add 250 µL of toluene and evaporate. Repeat 5×.
11. Place tube on ice and add 100 µL of saturated sodium bicarbonate solution.
12. Add 20 µL of acetic anhydride.
13. Mix gently and leave at 4°C for 10 min.
14. Add a further 20 µL acetic anhydride.
15. Incubate at room temperature for 50 min.
16. Pass solution through a column of Dowex AG50 X12 (H + form)—0.5 mL bed volume.
17. Wash tube with 4 × 0.5 mL of water and pass through a Dowex column.
18. Evaporate solution to dryness. This should be done in stages by redissolving in decreasing
volumes of water.
19. Prepare 50 × 2.5 cm strips of Whatman no. 1 chromatography paper (prewashed in water
by descending chromatography for 2 d).
20. Spot sample on strip.
21. Perform descending paper chromatography for 2 d in 441 by vol butanol–ethanol–
water for N-glycans and 821 1 by vol butanol–ethanol–water for O-glycans.
22. Remove strip from tank and allow all traces of solvent to evaporate.
23. Cut out region of strip from –2 cm to +5 cm from application.
24. Roll up cut chromatography paper and place in a 2.5-mL nonlubricated syringe.
25. Fit a 0.45 µm PTFE filter to syringe.
26. Add 0.5 mL of water and allow to soak into paper for 15 min.
27. Fit syringe plunger and force solution through filter.
28. Wash filter with water 4×, and pass through filter.
29. Evaporate sample to dryness, dissolve in 50 µL of water, transfer to microcentrifuge tubes,
and store at –20°C until required.