Waters GlycoWorks High-throughput Sample Preparation Kit - Manual (Page 2)

GlycoWorks High-throughput Sample Preparation Kit 2
[ CARE AND USE MANUAL ]
Table 1: Kit Components
Kit Component Purpose
GlycoWorks HILIC
µElution Plate Extract and clean up of free or FLR
labeled glycans
■ Capture of glycans
■ Removing contaminants like salt and
detergents from hydrophilic analytes,
such as carbohydrates, prior to MS
■ Removing excess
labeling reagent
■ The 5 mg sorbent was test to have
maximum binding capacity for 200 µg
glycans
GlycoWorks Glycoprotein
Control Standard Unlabeled IgG (100 µg/lyophilized) to
use as a control for process validation and
troubleshooting
RapiGest SF An enzyme friendly detergent used
to denature the glycoproteins prior to
deglycosylation reaction. For stability
purposes, there is enough RapiGest included
for initial use of the kit with 5 samples, more
may need to be ordered for processing all 46
samples.
Manifold Waste Tray For collection of non-releasing glycans
steps. This is disposable but can be
cleaned and used several times before
acquiring a new one as long as it is rinsed
out.
GlycoWorks Reagent Kit Contains ready to use reagents for both
denaturation, reduction, and alkylation as
well as raw materials for FLR tagging.
II. STORAGE AND DISPOSAL OF PLATES
Plates stored in their original sealed pouch remain stable for long
periods. To store unused plates in open pouches, squeeze the air out
of the pouch, fold over the open end of the pouch twice, seal with
tape, and store in a desiccator.
Note: Dispose of used plates safely in accordance with applicable
government or local regulations
III. ABBREVIATIONS
DMSO Dimethylsulfoxide IAM Iodoacetamide
DTT Dithiothreitol 2AB 2-aminobenzamide
IV. USING THE GLYCOWORKS HIGH-THROUGHPUT KIT
Below, are general guidelines for utilizing the GlycoWorks High-
throughput Kit for complete extraction, purification and concentration
of N-linked glycans validated using IgG. Also included is a check list of
accessory materials that will be needed to complete the protocol from
start to finish as well as tips and tricks throughout the instructions.
Table 2: Checklist of Additional Materials Needed Before You Begin
Material
Description
Recommended
Suppliers
Check List:
Circle
Ammonium
bicarbonate Sigma Aldrich
Cat No. O9830
Yes No
Ammonium acetate
Sigma Aldrich
Cat No. 73594 FLUKA
Yes No
Formic acid (to make
1% [v/v] formic acid,
prepared in 50%
acetonitrile)
Sigma Aldrich
Cat No. F0507
Yes No
15,000 units
(500,000 units/mL)
PNGase F*
New England Biolabs
Cat. No. P0704S
Yes No
Acetonitrile
Sigma Aldrich
Cat No. 360457
Yes No
25 mM ammonium
bicarbonate in water,
pH 8–9
See Above Yes No
100 mM ammonium
acetate in 5% [v/v]
acetonitrile, pH 7
See Above Yes No
85% [v/v] acetonitrile See Above Yes No
96-well collection plate
Waters P/N
186002481
Yes No
2-AB** Sigma P/N A89804 Yes No
µElution plate
manifold Waters P/N
186001831
Yes No
SPE vacuum pump
115, 60 Hz:
Waters P/N 176002986
Yes No
Positive pressure
manifold Waters P/N
186006961
Yes No
Centrifugal vacuum
evaporator
N/A Yes No
Heat block N/A Yes No
* PNGase F comparison table: https://www.neb.com/tools-and-resources/
usage-guidelines/glycobiology-unit-conversion-chart
**Inclusive Kits are also offered by ProZyme, Ludger, and Sigma covered
under US Patent No. 5,747,347 and its foreign equivalents.