Waters GlycoWorks High-throughput Sample Preparation Kit - Manual (Page 4)

GlycoWorks High-throughput Sample Preparation Kit 4
[ CARE AND USE MANUAL ]
Step 2b. Formic Acid Treatment of Released Glycans
1) Prepare 1% formic acid by adding 10 μL formic acid to 990 μL of
50% acetonitrile.
2) Add 50 μL of 1% formic acid solution to each glycan sample.
3) Incubate for 40 minutes at room temperature.
4) Dry glycans using vacuum evaporation bringing them to
complete dryness.
Step 3. Labeling of Glycans
Reference: Legal Information
Labeling of glycans with 2-AB is covered under US Patent No. 5,747,347
and its foreign equivalents.
Below is only an example of a sample procedure that one can follow
for labeling. Acetic Acid, DMSO, and Sodium cyanoborahydride are
included in the GlycoWorks Reagent Kit box labeled “Additional
Reagents” for your convenience in following this protocol with the
control standard.
Note: Prepare labeling solution immediately prior to labeling.
1) Add 300 μL of acetic acid vial to 700 μL of DMSO vial in the kit.
2) Weigh out 10 mg of 2AB.
3) Add 800 μL of the acetic acid/DMSO mixture to the entire vial of
chosen label.
4) Mix until dissolved (may require vortexing).
5) Add the entire contents of the labeled mixture to the vial of sodium
cyanoborohydride in the kit and mix until completely dissolved.
6) Add 10 μL of the labeling solution to each dried glycan sample.
Ensure glycans are fully reconstituted in the 2AB label.
7) Incubate glycan samples for a minimum of 3 h at 65 °C in a heating
block (if samples are in eppendorfs), dry oven, or sand tray.
8) Following incubation, briefly spin the vials to recollect each sample
at the bottom of the vial if samples are in Eppendorfs.
9) Allow samples to cool to room temperature.
Step 2 Tips and Tricks
■
The concentration of ammonium acetate and acetonitrile in
the elution buffers of Steps 2a and 4 may require optimization for
some glycans.
■ The reason for using low concentrations of formic acid is to
convert all glycans to free reducing glycans, hence improving
the overall yield of the FLR-labeling via reductive amination.
■ Drying down the sample does cause some sample loss but it
essential to have the sample completely dry before proceeding
to Step 3.
Step 3 Tips and Tricks
■
Protect the final preparation of labeling reagent from light.
■
The sodium cyanoborohydride once it is solubilized, should be
used within an hour.
■
Left over reagent which contains sodium cyanoborohydride has
to be disposed separately in a clearly marked bottle due to its
toxicity (see MSDS).
■
To ensure high yield of labeled glycans, it may be necessary,
for some samples, to increase the concentrations of the
reducing and labeling reagents.
General Guidelines for Vacuum Settings
■
For loading, washing, and elution a setting of 1–2 in Hg is
suggested.
■
For conditioning steps higher vacuum can be employed.
■
If no flow is observed, there may be an air bubble. Employ
high vacuum to initiate flow then return to an appropriate
vacuum setting.
General Guidelines for Positive Pressure Manifold:
■
For loading, washing, and elution, approximately 3 psi is
appropriate.
■
For conditioning, 10–20 psi can be used.