Waters GlycoWorks High-throughput Sample Preparation Kit - Manual (Page 5)

GlycoWorks High-throughput Sample Preparation Kit 5
[ CARE AND USE MANUAL ]
Step 4. Removal of Excess Label
1) Condition a NEW well of the Glycoworks μElution plate by add-
ing 200 μL of Milli-Q water to each well and aspirate using the
vacuum manifold or positive pressure manifold.
2) Add 200 μL of 85% acetonitrile to each well and aspirate using
the vacuum manifold or positive pressure manifold.
3) Add 100 μL of acetonitrile to the 10 μL of 2AB labeled glycan
sample and load into appropriate well on GlycoWorks HILIC
μElution plate using low vacuum setting (1-2 in Hg) or positive
pressure manifold.
4) Wash each well with 200 μL of 85% acetonitrile. Remove
filtrate to waste.
5) Repeat the washing step two more times.
6) Replace the waste tray with a 96-well collection plate.
7) Elute glycans with 50 μL of 100 mM ammonium acetate in 5%
acetonitrile.
Note: The concentration of ammonium acetate and acetonitrile in the
elution buffer may require optimization for some glycan samples.
8) Repeat elution two more times.
9) Place 96-well collection plate in a centrifugal vacuum evaporator
and dry the glycans to a concentrate of ≤ 1 µL in volume.
Note: If a rotor is not available for plates, transfer each eluent to a sterile
1.5 (or 0.5) mL eppendorf and place in the vacuum evaporator. This may
contribute to a small sample loss due to extra transfer.
10) Glycans can be stored in Milli-Q water at -20 °C until required.
V. PREPARING LABELED GLYCANS FOR HILIC-FLR WITH
AN ACQUITY UPLC BEH GLYCAN COLUMN
The following steps are suggested for preparing the labeled glycans for
HILIC-FLR with an ACQUITY UPLC BEH GLYCAN column when using this
kit with the Waters Total Glycan Application Solution.
1. Reconstitute the labeled glycans in 50 µL of Milli-Q water by
gently aspirating, avoiding vortexing and sonication.
2. Add 75 µL of ACN to the reconstituted glycans immediately prior
to and in preparation for analysis by HILIC-FLR.
3. Inject 4 µL of the labeled glycan containing 60% ACN solution
onto the column.
Note: Preparations of non-IgG glycoproteins or glycoprotein samples with
concentrations other than 1 mg/mL may need to be reconstituted with
different volumes to achieve desired results. Similarly, more concentrated
samples can be obtained by reconstituting with up to 10 times lower volumes.
The optimum injection volume for this application is ≤4 µL.
VI. REFERENCES
1. Lauber MA, Koza S, Fountain KJ. Optimization of HILIC SPE for the Quantita-
tive and Robust Recovery of N-Linked Glycans. Waters Application Note
720004710EN. 2013 June.
2. Lauber MA, Koza S, Fountain KJ.Single-Use and High-Throughput HILIC SPE
Device Formats and an IgG Control Standard for Facilitating N-Glycan Analy-
ses Waters Technology Brief 720004711EN. 2013 June.
3. Training Video: http://www.waters.com/waters/en_US/Glycan-Standards/nav.
htm?cid=134640534
Step 4 Tips and Tricks
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Extended periods of time between incubation and analysis
may result in desialylation of labeled glycans and consequently
should be avoided.