FACE® N-Linked Oligosaccharide Profiling Kit - ProZyme

6 Add 2 µl of N-Glycanase (C1) to the glycoprotein sample and Oligo Profiling Control (E4). Mix
with finger flicks and centrifuge for 5 seconds. Store any remaining enzyme at 4ºC.
7 Incubate for 2 hours or overnight at 37°C.
8 Prepare the OLIGO Quantitation Control (E5):
a. Add 300 µl of distilled water to the tube labeled E5, which contains 6 nmoles of maltotetraose.
b. Resuspend the lyophilized carbohydrate by vortexing.
c. The concentration of the maltotetraose is now 200 pmoles/10 µl.
9 Add 10 µl (200 pmoles) of reconstituted E5 to the glycoprotein digest as an internal labeling control,
or label 10 µl in a separate tube to check your quantitation according to the guidelines on page 4.
Store the unused reconstituted E5 at -20°C.
10 Precipitate the protein by adding 3 volumes of cold ethanol. Keep samples on ice for 10 minutes.
Spin samples in microcentrifuge for 5 minutes to pellet the protein.
11 Remove the supernatant and transfer to a clean 1.5 ml microcentrifuge tube.
DO NOT DISCARD THE SUPERNATANT; the released carbohydrates are present in the
supernatant.
12 If a large amount of protein was digested (>250 µg), 5-10% of the released oligosaccharides may
remain in the pellet. The recovery of these oligosaccharides can be accomplished by drying the
pellet completely in a centrifugal vacuum evaporator or lyophilizer. Add 50 µl water to resuspend,
then 150 µl of cold ethanol and precipitate on ice. Centrifuge and combine the supernatants.
13 Dry supernatants in a centrifugal vacuum evaporator or lyophilize to a translucent pellet.
At this point samples may be stored at -20°C, or proceed with the fluorophore labeling procedure
described in Section 2.
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