GlykoPrep - plus Rapid N-Glycan Sample Preparation with 2 ......Labware Set from ProZyme (product...
Transcript of GlykoPrep - plus Rapid N-Glycan Sample Preparation with 2 ......Labware Set from ProZyme (product...
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GlykoPrep -plus Rapid N-Glycan Sample Preparation with 2-AB Automated on AssayMAP Technology®
An automated, highly robust, high-throughput process for enzymatic deglycosylation, fluorescent labeling with 2-AB (2-aminobenzamide) and cleanupof excess dye for analysis by LC and other methods.
� Non-selective, rapid release and recovery of N-glycans from up to 96 glycoprotein samples at a time on the Agilent AssayMAP Bravo LiquidHandling Workstation (AssayMAP Bravo)
� Minimal hands-on time
� Optimized reaction conditions help to preserve labile glycans, such as sialic acid
� Non-selective chemistry for stoichiometric labeling of glycans, independent of structure
� Complete labeling in 1 hour using Rapid-Reductive-Amination™
� Purified 2-AB-labeled N-glycans are eluted in water, ready for analysis
Product Code: GPPNG-AB
NOTICE: ProZyme was purchased by Agilent in July 2018. Documents for products and product lots manufactured before August 2019 will contain references to ProZyme. For more information about these products and support, go to: www.agilent.com/en/contact-us.
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TABLE OF CONTENTS
Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
How to Use This Booklet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
GPPNG-AB Kit Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Storage Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Additional Required Reagents/Equipment. . . . . . . . . . . . . . . . . . . . . . . . 5Optional Reagents and Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Safety and Handling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Using the Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6GlycoProtein Sample Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Using the Reagent Volume Calculator.. . . . . . . . . . . . . . . . . . . . . . . . . . 10GlykoPrep-plus Reagent Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . 11AssayMAP Bravo Processing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Preparing for the GlykoPrep-plus Protocols. . . . . . . . . . . . . . . . . . . . . . 13
Glycoprotein Sample Plate Preparation. . . . . . . . . . . . . . . . . . . . . . 13Reagent Source Plate Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . 13Optional Labeling Source Plate Preparation. . . . . . . . . . . . . . . . . . . 14Preparation of the AssayMAP Bravo and Associated Equipment. . . 14
GlykoPrep-plus Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Plate & Reagent Setup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . 162 RX Cartridge Setup Protocol.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Immobilization & Digestion Protocol. . . . . . . . . . . . . . . . . . . . . . . 20Manual Processing - Drying the N-Glycans.. . . . . . . . . . . . . . . . . . . 234 Cleanup Cartridge Setup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . 24Manual Processing - Labeling the N-Glycans. . . . . . . . . . . . . . . . . . 265 Labeling Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 Cleanup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
QuickGuide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Analysis of Labeled Glycans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Tips & Hints. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Technical Assistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
License to Use.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Trademarks & Tradenames. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Product Use and Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Other ProZyme Products & Kits. . . . . . . . . . . . . . . . . . . . . . . . . 47
Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
This product is intended for in vitro research use only.
NOTE: The following suggestions and data are based on inform ation we believe to be reliable. They
are offered in good faith, but without guarantee, as conditions and methods of use of our products are
beyond our control. We recom m end that the prospective user determ ine the suitability of our materials
and suggestions before adopting them on a com m ercial scale. Suggestions for use of our products or
the inclusion of descriptive material from patents and the citation of specific patents in this publication
should not be understood as recom m ending the use of our products in violation of any patent or as
perm ission to license to use any patents of ProZym e, Inc.
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EQUIPMENT
The AssayMAP Bravo and associated equipment are available from Agilent Technologies.
Comments
AssayMAP Bravo Liquid Handling Workstation (AssayMAP Bravo, p/n G5542A) Bravo 96 AM Head (p/n G5498B#046)
Peristaltic Pump Module 2.0 (p/n G5498B#058) linked to 96AM Wash Station (p/nG5498B#057)
Peltier Thermal Station - with STC Controller (p/n G5498B#035)Cartridge Seating Station (aka Tips Loading Station)Orbital Shaking Station w/ Control Unit (p/n G5498B#033)96AM Cartridge & Tip Loading Station (p/n G5409-20025)Gripper upgrade (p/n 16545-101)Risers, 146 mm (p/n G5498B#055)Custom Plate Nest (p/n G5498B#017)PCR Plate Insert (p/n G5498B#013)Carboy to feed pump & collect waste ( 2 ea, G5550-17725)Lab tape (to tape the wash station, RX/CU Cartridge Racks and Receiver Plate to the
deck)
SOFTWARE
Agilent VWorks "GlykoPrep-plus Full App version 11.2.0.1182" or newerInstaller with N-Glycan Sample Prep: RX digestion & 2-AB labeling v1.0 or newer
HOW TO USE THIS BOOKLET
We want successful results for our customers, so please read this entire bookletbefore starting the procedure, and follow the detailed instructions.
Once familiar with the AssayMAP Bravo and GlykoPrep-plus protocols, experiencedusers may wish to use the streamlined QuickGuide section beginning page 33.
AssayMAP Bravo must be installed, calibrated, and set upwith the GlykoPrep layout and recommended software.
Print the QuickGuide separately, using PortraitOrientation.
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KIT CONTENTS
GPPNG-AB Kit GlykoPrep-plus Rapid N-Glycan Sample Preparation with 2-AB
GSP-RX GlykoPrep-plus Digestion ModuleWS0253 RX CartridgesWS0256-GP Immobilization Reagent Set
WS0226-GP Denaturation Reagent (35 ml)WS0255-GP Blocking Reagent (14 ml)
WS0259-GP Digestion Reagent SetWS0229-GP Finishing Reagent (10 ml)WS0276-GP 25x Digestion Buffer (1.3 ml)WS0278-GP N-Glycanase (700 �l)
GS96-AB GlykoPrep Rapid-Reductive-Amination 2-AB Labeling Module WS0302 Rapid 2-AB Solution (325 �l) WS0300 Reductant Solution (325 �l)
GSP-CU GlykoPrep-plus Cleanup ModuleWS0263 CU Cartridges (96)WS0303-GP 5x 2-AB Sample Load Solution (12 ml)Aluminum Sealing Film (2)
Labware Set from ProZyme (product code AM96-NG when purchased alone)WS0304 1 ea, 12-Column, Reservoir Plate (5-pack)WS0305 4 ea, PCR PlateWS0306 2 ea, Square, Deep-Well PlateWS0307 1 ea, Pipette-Tip BoxWS0308 1 ea, U-Bottom Plate (10-pack)
Aluminum Sealing Film (2)
1 ea
1 ea
1 ea
1 ea
Confirm all materials are present before beginning.
Ships with Product Code GPPNG-AB. These are thenames used throughout the protocols.
WARNING: The supplied protocols and applicationsupport a specific list of labware, which is included inthe purchase of the GPPNG-AB Kit. If labware otherthan those listed here are to be used, the protocol andapp require editing. Using undefined or incorrectlyidentified labware on the AssayMAP Bravo deck cancause damage to the Bravo 96AM Head.
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Storage Requirements
This Kit is a mixed-temperature shipment (2–30�C). Upon arrival, storecomponents as indicated. For best results, equilibrate materials to ambienttemperature prior to use. The 2-AB Solution and Reductant Solution arehygroscopic and the 2-AB Solution is light-sensitive; please store these reagentsat -20�C in their original packaging.
Additional Required Reagents/Equipment
Ultrapure, deionized water (~100 ml, Milli-Q or equivalent)®
100% Acetonitrile (~250 ml, HPLC-grade)Microplate-compatible, centrifugal evaporator (e.g., SpeedVac with plate®
rotor or similar)Fume hoodVortexerNitrile GlovesCalibrated pipettes and disposable tips (P5/10, P200 and P1000)
Volumetric Pipettes (5-ml, 1 ea)Glass Graduated Cylinder (50-ml, 1 ea)Glass Bottle with Screw Cap (50-ml, 1 ea)
Optional Reagents and Supplies
Multichannel pipettors & disposable tips (P5/P10 and P200) (Gilson orequivalent, compatible with Gilson Diamond D200 pipette tips)®
SAFETY AND HANDLING
Some of the reagents in this Kit are hazardous. Please refer to the Safety DataSheets (SDS) included with the Kit or posted on ProZyme’s website under thecomponent name or Product Code.
http://www.prozyme.com
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General Laboratory Procedures
Use powder-free, nitrile gloves for all sample handling procedures. Ensure thatall glass, plasticware and solvents are free of glycosidases and environmentalcarbohydrates.
All procedures involving Labeling Reagents (2-AB Solution, Reductant Solutionand 2-AB Labeling Reagent) should be performed in a dry environment withdry glassware and plasticware, using appropriate personal safety protection,eyeglasses and nitrile gloves, and where appropriate, in a fume hood.
INTRODUCTION
The GlykoPrep-plus Sample Preparation Platform (GlykoPrep-plus) dramaticallystreamlines glycoanalysis by automating deglycosylation and separation ofN-glycans, complete fluorescent labeling and efficient cleanup to reduce excessreagent peaks using the Bravo 96AM Syringe Head on the AssayMAP Bravo.
GlykoPrep-plus is modular and can be integrated into any workflow, regardlessof throughput or sample type. In order to match any standard samplepreparation, Kit components may be made available individually; for moreinformation please contact us.
GlykoPrep-plus is built on AssayMAP technology, microchromatography in a96-well format using the Syringe Head on the Agilent AssayMAP Bravo tomove liquid through the Cartridges. The same procedures may be performedusing centrifugation (GlykoPrep, spin format). For more information aboutGlykoPrep, please visit our website:
http://www.prozyme.com/GlykoPrep/
USING THE KIT
GlykoPrep-plus Rapid N-Glycan Preparation with 2-AB combines the DigestionModule, the Rapid-Reduction-Amination 2-AB Labeling Module and theCleanup Module, which may be purchased individually. The Labeling andCleanup Modules may be purchased together as GPP-ABCU.
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This GPPNG-AB Instruction Manual provides instructions for use of the AssayMAPBravo for N-glycan processing using GlykoPrep-plus Rapid N-Glycan SamplePreparation with 2-AB Kit (GPPNG-AB). It also provides additional backgroundinformation and explanations to frequently-asked questions regarding:
• Glycoprotein Sample Preparation• Using the Reagent Volume Calculator • AssayMAP Bravo Processing Time• GlykoPrep-plus Reagent Preparation
Glycoprotein Sample Preparation
This section discusses a number of considerations when preparing GlycoproteinSamples for processing on the AssayMAP Bravo.
� Optional Purification
The GPPNG-AB Kit begins with purified Glycoprotein Samples. To process cellculture or other mixtures in a single workflow, a purification module, such asProtein A or Protein G, may be employed just prior to Protocol 1 on the AssayMAPBravo.
Glycoprotein Samples must not contain any particulates, as they will plug the topfrit, or sit on the top of the resin bed and impede the flow. Spin samples to removeparticulates before processing.
� Sample Quantities
The quantitative binding capacity for each Cartridge is:
RX Cartridge 50 �g of any standard proteinCU Cartridge 30 �g of N-glycans
The binding capacity for specific glycoproteins may needto be determined.
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Each Cartridge is capable of binding more target, but will do so with increasingbreakthrough, making the process non-quantitative. Less than the maximum quantitymay be processed, for example, when the sample is available only in limited amounts. The smallest effective amount of sample will depend on the sensitivity requirements ofthe analytical methods used and the specific application.
� Sample Concentrations
For standard processing, the Glycoprotein Sample concentration should be in therange of 1–5 mg/ml, and sufficient reagents have been provided to processindividual columns (8 samples) at a time. The GPPNG-AB Kit can also be used formore dilute samples (down to 0.05 mg/ml); the effective minimum SampleConcentration will depend on the denaturation requirements for the specificglycoprotein (see below).
If quantitation is desired, pipetting less than 10 �l is notrecommended; pipetting smaller volumes introducesvariability, especially when samples are highlyconcentrated. If necessary, dilute the sample to withinthe 1-5 mg/ml range with Digestion Buffer beforebeginning.
NOTE: All Glycoprotein Samples will be loaded at thesame volume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.
� Sample Denaturation
Prior to the enzyme digest, the Glycoprotein Samples are denatured by pre-mixingwith Denaturation Reagent to open up the protein structure and allow access to thedeglycosylation enzyme. The standard protocol employs a 5-minute, relativelygentle denaturation; the Denaturation Reagent is 6 M Guanidine. Start with a 1:1ratio of Glycoprotein Sample to Denaturation Reagent, and increase the ratio ofDenaturation Reagent to 9:1 to test the effects of increasing level of DenaturationReagent. Most glycoproteins can be adequately denatured under these conditions.
The amount of Denaturation Reagent to be added isdetermined by setting the Denaturation Factor in theReagent Volume Calculator (see instructions page 11).
Any custom denaturation may be performed prior toprocessing on the AssayMAP Bravo, as long as no SDS orother detergents are used.
In all cases, a minimum of 50% Denaturation Reagent should be added prior toloading onto the RX Cartridge. This assures that the Glycoprotein Sampleformulation matches the equilibration conditions of the RX Cartridge, and avoidsprecipitation of the protein, which will plug the Cartridge.
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� Deglycosylation Temperature
The assay has been optimized for a setting of 45�C. Note that this temperature is theset point, and not the actual reaction temperature, which is a few degrees lower.
The user may enter temperatures other than the recommended setting forqualification or optimization studies.
� Duration of the Deglycosylation Reaction
The digestion procedure has been optimized to deliver deglycosylation of N-Glycansfrom most glycoproteins in 15–60 minutes. The optimal incubation time will varydepending on the specific glycoprotein; those which have proven to be resistant todeglycosylation via conventional enzymatic methods may require longer incubationtimes (up to 60 minutes). For glycoproteins that are comparatively easy todeglycosylate, such as monoclonal antibodies, a 15-minute incubation is generallysufficient.
Often glycoproteins must be denatured to open the protein structure for the enzymeto gain access for cleavage. See the discussion in this section under SampleDenaturation.
It is critical not to exceed a 60-minute incubation, as theCartridge resin bed may dry out, yielding uncertainresults.
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Using the Reagent Volume Calculator Excel spreadsheet file that uses formulas to calculatereagent needs and recipe volumes.
Reagents and other solutions, either provided by the user or supplied with theGlykoPrep-plus Kit, must be prepared prior to dispensing into the Reagent SourcePlate.
NOTE: Some values incorporate a small overagerequired for proper operation of the probes. Forexample, for Required Sample Volume and DenaturationReagent Volume, the calculator automatically adds 5 �l.
Enter the sample information in the green fields at the top of the Sample Info &Reagent Prep tab. Grey fields are calculated values.
Volumes listed in the Reagent Source Plate tab andoptional Labeling Source Plate tab are based on numbersentered into this tab.
� Number of Samples: _________ (actual, green)
Enter the total number of glycoprotein Samples to be processed.
This field is used to calculate the number of columns andvolume of reagents required. Normal processing is donewith complete plate columns, so the number of Samplesshould be 8 to 96 and divisible by 8.
� Number of plate columns used: (calculated value, grey) This is the Number of Samples divided by 8, as only fullcolumns are used. Used to calculate the amount ofreagents to be prepared.
� Target Load (�g): _________(actual, green)
Enter the desired Glycoprotein Sample Target Load.
The maximum, quantitative Glycoprotein Sample TargetLoad on the RX Cartridge is 50 �g. This value is usedwith the Glycoprotein Sample Concentration to calculatethe total Denatured Sample Load Volume.
� Sample Concentration (mg/ml): _________(actual, green)
Enter the concentration of the Glycoprotein Samples.
All Glycoprotein Samples will be loaded at the samevolume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.
� Required Starting Sample Volume (�l): (calculated value, grey) Calculated from the Sample Target Load and SampleConcentration. Dispense this volume accurately into theGlycoprotein Sample Plate (page 13). This cell will turnred for values greater than 250 �l as this is outside thevolume limit of this parameter.
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� Denaturation Reagent Factor (x:1): _________(actual, green)
Enter the ratio of Denaturation Reagent to be added to the GlycoproteinSamples; the Denaturation Reagent Factor is the x in the relationship (X:1).
For example, recommended to start: enter 1 for a 1:1mixture to achieve 50% Denaturation Reagent.
� Denaturation Reagent Volume (�l): (calculated value, grey) Calculated from previous entries. This cell will turn redfor values greater than 200 �l, as this is outside thevolume limit of this parameter.
� Denatured Sample Load Volume (�l): (calculated value, grey) Sum of the Glycoprotein Sample Volume and theDenaturation Reagent Volume, less 10 �l to ensureproper filling of the probes.
NOTE: If the sum of the Required Starting SampleVolume plus the Denaturation Reagent Volume less 10 �lis greater than 250 �l, this cell will turn yellow and thevalue will appear as 250 �l, which is the volume limit ofthe syringe.
GlykoPrep-plus Reagent Preparation
Reagents and other solutions, either provided by the user or supplied with theGPPNG-AB Kit, must be prepared prior to dispensing into the Reagent Source Plate. Please refer to the directions in the GPPNG-AB Reagent Volume Calculator (Excel files)to determine the appropriate amount of each to prepare. The “Sample Info and ReagentPrep” tab and the “Reagent Source Plate Setup” tab of the Reagent Volume Calculatormay be printed and taken to a laboratory work station to prepare and then dispensereagents and solutions as shown. The AssayMAP Bravo will dispense the reagents andsolutions from the Reagent Source Plate to begin the sample preparation.
The amount of N-Glycanase (700 �l plus overfill) provided in the kit is sufficient for thepreparation of 96 samples, in a single run or in 4 runs totaling 96 replicates. If smallerruns are desired, it is important to refer to the reagent calculator to confirm the amountof enzyme needed. Additional enzyme is needed to perform greater than 4 runs with asingle kit; additional N-Glycanase (WS0278-GP) can be purchased from ProZyme.
See Preparing for the GlykoPrep-plus Protocols andGlykoPrep-plus Protocols pages 13-32 for preparation ofspecific reagents.
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The labware provided in the kit is sufficient for a single run of up to 96 samples. Ifmultiple runs are desired, additional labware may be purchased as a kit (AM96-NG) or inbulk from ProZyme. Please contact ProZyme for bulk purchase inquiries.
GPPNG-AB Kit components are shipped at concentrations that assure maximum stability;some will be diluted prior to dispensing and others will be dispensed directly from thekit containers.
NOTE: Equilibrate all reagents to room temperature, thengently invert to mix.
AssayMAP Bravo Processing Time
The approximate time to process Glycoprotein Samples on the AssayMAP Bravodepends on the specific choices when entering values in the various protocols. Theapproximate times for standard processing are shown in Table 1:
Protocol Fixed Variable Total
(min)The Variable times listed in Table 1 may be influenced bythe following factors:
• Number of columns in use• Digestion incubation time• Denaturation incubation time• Drying time• Load (up to 250 �l at 5 �l/min)• Manual pipetting efficiency• Preheating of the heater before the labeling step
(saves 10 minutes)
1. Plate and Reagent Setup Protocol 15 15 15 - 30
2. RX Cartridge Setup Protocol 1 n/a 1
3. Immobilization and Digestion Protocol 90 30 90 - 120
Drying N-Glycans Prior to Labeling 20 40 20 - 60
4. CU Cartridge Setup Protocol 1 n/a 1
Labeling the N-Glycans with 2-AB n/a 5 - 15 5 - 15
5. Labeling Protocol 70 10 70 - 80
6. Cleanup Protocol 35 n/a 35
Total 237 - 342
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Preparing for the GlykoPrep-plus Protocols
Glycoprotein Sample Plate Preparation
Prepare the Glycoprotein Sample Plate with required volumes as described forprocessing on the AssayMAP Bravo.
See: 1. Sample Info & Reagent Prep tab in the Reagent
Volume Calculator to compute starting volumes. 2. “GlykoPrep Sample Preparation” above for
information about Glycoprotein Sample concentrationand denaturation conditions.
Dispense the Glycoprotein Samples into either a PCR Plate or a U-Bottom Platedepending on the final Denatured Glycoprotein Sample Volume.
Use the PCR Plate if the Total Volume is 110 μL or less;use the U-Bottom Plate if the Total Volume is110 to 300 μL.
NOTE: Be sure not to introduce bubbles; spin down ifnecessary
NOTE: All Glycoprotein Samples will be loaded at thesame volume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.
Reagent Source Plate Preparation
� Print the “Sample Info & Reagent Setup” and “Reagent Source Plate Setup” tabsof the Reagent Volume Calculator.
� Prepare the reagents and solutions needed as described below and in the greysection of the “Sample Info & Reagent Setup” tab.
NOTE: The color cues used here match the protocolcolors in the Agilent application interface.
1. Digestion Buffer
In a separate vial, add the amounts of 25x Digestion Buffer and ultrapure wateras indicated in the Reagent Volume Calculator, and then vortex to mixthoroughly.
� Digestion Buffer may be prepared up to one weekbefore use. Store at 2–8�C.
2. Enzyme Solution
Vortex to mix the vial of N-Glycanase, and then spin down briefly to collect thecontents in the base of the vial.
In a separate vial, add the amounts of Digestion Buffer (prepared above) andN-Glycanase indicated in the Reagent Volume Calculator, and then vortex to mix
� Enzyme Solution should be prepared on the day ofuse; store at RT.
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thoroughly.
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3. Blocking Reagent
4. Denaturation Reagent
5. Finishing Reagent
Pipette prepared solutions from the previous step into each well location in theReagent Source Plate as shown in the table in the “Reagent Source Plate Setup”tab.
NOTE: Be sure not to introduce bubbles; spin down ifnecessary
Optional Labeling Source Plate Preparation
No more than one hour prior to use, Labeling Reagent may be prepared inColumn 1 of the Labeling Source Plate and transferred manually by multi-channel pipette to the N-glycan Samples in the Processing Plate.
For preparation of Labeling Reagent, see ManualProcessing - Labeling the N-Glycans with 2-AB, page 26and Labeling Source Plate tab of the Reagent VolumeCalculator.
Preparation of the AssayMAP Bravo and Associated Equipment
Turn on the AssayMAP Bravo, computer, pump and Inheco controller.
Open the AssayMAP LaunchPad from the desktop.
Under the N-Glycan Sample Prep tab, click on the 2-AB icon to launch N-GlycanSample Prep: RX digestion & 2-AB labeling.
Verify that the Wash Station is installed on position#1.
� Connect appropriate tubing from the DI water container to the pump, from thepump to the wash station, and from the wash station to the waste container.
� Fill the wash station source container with DI water (if necessary).� Empty the wash station waste container (if necessary).� Click on “Prime and Wash” to prime the chimneys.� Verify that all chimneys have water flowing.
Verify that the Peltier Thermal Station with PCR Plate Adapter is installed onpostion#4.
Verify that the Orbital Shaking Station is installed on position#9.
Use the LaunchPad web interface to access the ReagentVolume Calculator, GlykoPrep-plus Protocols andsupport documents
NOTE: Ensure that the lines are flushed if changingcomposition of the source.
5 L of purified, deionized water is needed.
During Prime and Wash, syringe tips are washed with 10volumes of DI water, sufficient to eliminate carryoverbetween steps.
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GLYKOPREP-PLUS PROTOCOLS
1. Plate & Reagent Setup Protocol (grey)
2. RX Cartridge Setup Protocol (green)
3. Immobilization and Digestion Protocol (blue)
4. CU Cartridge Setup Protocol (purple)
5. Labeling Protocol (orange)
6. Cleanup Protocol (red)
In this section, the colored tables give a Deck Layout, aLabware Table and Application Settings for each of theAssayMAP Protocols.
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1 Plate & Reagent Setup ProtocolAssumes full columns are processed
Set the parameters in the Application Settings section:1. Number of Columns to Manage (columns of 8 samples) = _________2. Starting Column of Destination Plate = _________
Check the previously prepared Reagent Source Plate for bubbles; centrifuge brieflyto clear bubbles if necessary.
Third section of the grey graphic above.
Normally Starting Column will be set to 1 unlesspreviously used plates are being reused. This setting willbe the same for all destination plates.
Place the Reagent Source Plate on position#5. From page 13, prepared using the Reagent VolumeCalculator.
Place the empty Seating Station on position#3.
Label a clean PCR Plate as “Processing Plate,” and place it on the Heater StationAdaptor on position#4.
The Processing Plate will contain the released N-glycansafter Immobilization and Digestion.
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Place a Pipette-Tip Box on position#6; REMOVE THE LID. If using less than a full box, the pipette tips must be onthe RIGHT side of the box as you look at the AssayMAPBravo from the front. This is counterintuitive, so pleasedouble check.
This protocol uses 40 pipette tips, which are thendiscarded. Make sure there are at least 40 tips in fullcolumns in the rack of pipette tips.
Label and stack 4 U-Bottom Plates:a. Digestion Buffer (top)b. Blocking Reagentc. Denaturation Reagentd. Finishing Reagent (bottom)
Place the stack on position#7.
Orient the plates so that the A1 wells are pointed towarddeck position#1.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the grey graphic above; labware is listedin the second section.
Click “Run Protocol 1.” A status line will show when the protocol is complete: "Run complete. Module idle."
Approximate run time is 15 minutes.
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
Proceed to Protocol 2. May delay as much as one hour without affecting resultsbefore proceeding to the next protocol.
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2 RX Cartridge Setup ProtocolAssumes full columns are processed
Rearrange the deck in preparation for processing:1. Remove the Reagent Source Plate from position#2; discard.2. Remove the Seating Station on position#3 and discard the used pipette tips.3. Place the now empty Seating Station on position#2.4. Remove the lid from a Rack of RX Cartridges and place it with its Receiver Plate
on position#6.5. Tape the Cartridge Rack and Receiver Plate to the deck.
Set the parameters in the Application Settings section:
1. Indicate the first column of RX Cartridges to be used in the RX Cartridge Rack inposition#6.
2. Indicate the number of columns to transfer.
Third section of the green graphic above.
NOTE: The first column to be used must be the firstavailable column of Cartridges as the Syringe Headcannot access a buried column of Cartridges. Remainingcolumns are transferred directly after the first column inthe left to right direction.
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3. Indicate the first column in the Seating Station into which these columns of RXCartridges will be placed.
Normally, the starting column of the RX Cartridge Rackwill match the starting column of the Seating Stationworking across a plate from left to right. For example,columns 1-5 in a run using a full rack would be indicatedby starting column #1 and 5 columns to be moved. Thesecond run using that rack would start with column 6.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the green graphic above; labware is listedin the second section.
Click on “Run 2 RX Cartridge Setup.” NOTE: The RX Cartridge Setup module can be reversedby clicking on “Undo RX Cartridge Transfer.”
A status line will show when the protocol is complete: “Run complete. Module idle.”
Approximate run time is 1 minute.
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
Proceed to Protocol 3. May delay as much as one hour before proceeding to thenext protocol without affecting results.
-
21
3 Immobilization & Digestion Protocol Assumes full columns are processed
Dispense the RX Priming Solution (100 % Acetonitrile) into a 12-Column ReservoirPlate. Fill the channels with the specified volume corresponding to the columns ofRX Cartridges to be processed.
Rearrange the deck in preparation for processing:
1. Remove the RX Cartridge Rack, Receiver Plate and tape from position#6. Replace the lid to protect any remaining RX Cartridges, and store appropriately.
2. Place the 12-Column, Reservoir Plate containing the RX Priming Solution onposition#3, making sure that the filled reservoirs correspond to the RX Cartridgepositions.
Blue section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab.
-
22
3. Place the Glycoprotein Sample Plate on position#6. The positions of the columns of Glycoprotein Samplesshould correspond to the positions of the columns of RXCartridges.
NOTE: The Glycoprotein Sample Plate can be a PCRPlate or a U-Bottom Plate, depending on the DenaturedSample Load Volume. Use a PCR Plate if this value is110 μL or less; use a U-Bottom Plate if it is between 110and 300 μL.
Set the sample plate type in the “Labware” section, line 6:Select PCR Plate (low volume) or U-Bottom Plate (high volume).
Middle section of the blue graphic above.
WARNING: The correct sample plate type MUST bechosen or the AssayMAP Bravo Head may be damaged.
Set the parameters in the Application Settings section: Third section of the blue graphic above.
1. Denaturant Volume: Enter the volume of Denaturation Reagent to be added toeach Glycoprotein Sample.
Cell D16 in the Reagent Volume Calculator. Themaximum allowable volume is 200 �l.
2. Starting Sample Volume: Enter the starting volume of Glycoprotein Sample to beloaded.
Cell D15 in the Reagent Volume Calculator. Themaximum allowable volume is 250 �l.
3. Denatured Sample Load Volume (calculated value, grey). The Denatured Sample Load Volume is calculatedautomatically (from cells D15 and D16 of the ReagentVolume Calculator, less 10 �l to ensure proper filling ofthe probes) and displayed on the form after the protocolbegins.
The maximum volume that can be loaded on a Cartridgeis 250 �l; if the sum of the Denaturant Volume and theStarting Sample Volume is greater than 260 �l, only 250 �lwill be loaded.
4. Enter the Sample Loading Flow Rate. The recommended flow rate is 5 �l/minute, althoughfaster or slower rates are possible. Flow rates other thanthe recommended rate of 5 �l/minute may affect themaximum quantitative binding capacity.
5. Temperature Setpoint for Digestion: Enter the incubation temperature for thedeglycosylation reaction.
Note that this temperature is the set point and not theactual reaction temperature, which is a few degreeslower. The assay has been optimized for a set point of
-
23
45�C, although laboratory conditions may cause thisoptimum to vary slightly.
The temperature can be monitored on the digital readoutof the STC Controller.
6. Duration of Digestion Step: Enter the duration for the deglycosylation reaction. The level of glycosylation varies from glycoprotein toglycoprotein; see “Duration of the DeglycosylationReaction” page 9.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout. First section of the blue graphic above; labware is listedin the second section.
Click on “Run 3 Immobilization & Digestion.” The protocol will take between 90 and 120 minutesdepending on the sample volume and incubation time.
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
A status line will show when the protocol is complete: “Run complete. Module idle.”
Proceed to Manual Processing. The Processing Plate (position#4) now contains theN-glycans; remove for drying. The AssayMAP Bravo maybe shutdown at this point.
-
24
Manual Processing - Drying the N-Glycans prior to labeling
Rearrange the deck in preparation for processing:
� Remove the Processing Plate (PCR plate) from position#4.
� Dry the N-Glycan Samples using a centrifugal evaporator with the heat settingturned to the off position for 20 minutes to 1 hour, or until fully dry. .
The Processing Plate now contains released N-Glycanswith a free reducing end, along with Finishing Reagent.
This is a good time to remove the 2-AB Solution andReductant Solution so they can come to roomtemperature.
Protocol 4 may be processed simultaneously with Manual Processing.
-
25
4 CU Cartridge Setup Protocol Assumes full columns are processed
Rearrange the deck in preparation for processing:� Remove all Immobilization and Digestion labware from deck.� Discard used RX Cartridges.
Remove the lid from a Rack of CU Cartridges and place it with its Receiver Plate onposition#6.
May process a partial Rack.
Tape the CU Cartridge Rack and Receiver Plate to the deck.
Place the empty Seating Station in position#2.
-
26
Set the parameters in the Application Settings section:
1. Indicate the first column from which to take the CU Cartridges from Rack inposition#6.
2. Indicate the number of columns to transfer.3. Indicate the first column in the Seating Station where the CU Cartridges are to be
placed.
Third section of the purple graphic above.
NOTE: This must be the first occupied column of the CUCartridge Rack as the Syringe Head cannot access aburied column of Cartridges.
Normally the starting column of the CU Cartridge Rackwill match the starting column of the Seating Stationacross the plate from left to right.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the purple graphic above; labware is listedin the second section.
Click on “Run 4 CU Cartridge Setup.” NOTE: the CU Cartridge Setup module can be reversedby clicking on “Undo CU Cartridge Transfer”.
A status line will show when the protocol is complete: “Run complete. Module idle.”
Approximate run time is 1 minute.
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
When the N-glycan Samples are dry, proceed to Manual Processing - Labeling theN-Glycans with 2-AB.
-
27
Manual Processing - Labeling the N-Glycans with 2-AB
Prepare Labeling Reagent.
.
Allow the 2-AB Solution and Reductant Solution vials to come to room temperaturein the sealed desiccant bag before removing them. Invert each to mix gently. Before opening each vial, flick it or gently tap it on a flat surface to dislodge anyliquid adhering to the underside of the cap and ensure that the contents collect atthe bottom.
In a separate vial, add the amounts of 2-AB Solution and Reductant Solution asindicated in the Reagent Volume Calculator, and then vortex to mix thoroughly.
Pipette equal volumes of 2-AB Labeling Reagent into each well of the first column ofthe Labeling Source Plate (PCR plate) as instructed in the Labeling Source Plate tabof the Reagent Volume Calculator.
Add the Labeling Reagent:� Add 5 μl of Labeling Reagent to the side of each N-Glycan Sample well about
half way down the side of the well.� Tap the plate sharply on the bench and centrifuge briefly to ensure that the
droplet of Labeling Reagent drops to the bottom of the well.� Cover the plate with the Aluminum Seal provided.
Orange section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab.
� Prepare no more than one hour before use
NOTE: 2-AB Labeling Reagent components arehazardous. Please refer to the Safety Data Sheetsincluded in the Kit or on our website. Perform thisprocedure using appropriate personal safety protection,eyeglasses and nitrile gloves.
NOTE: Both the 2-AB Solution and Reductant Solutionare hygroscopic; minimize exposure to air and protectfrom exposure to light. The individual reagents may betightly capped, repackaged with the desiccant in theresealable bag, and frozen (-20�C) for storage up to 6months.
Use of the Labeling Source Plate is optional.
Dried N-glycans in Processing Plate
Visually inspect to make sure there are no bubbles at thebottom of the well.
Remove the tabs from the Aluminum Seal, as they caninterfere with operation of the AssayMAP Bravo.
Proceed to Protocol 5.
-
28
5 Labeling Protocol
Place the sealed Processing Plate containing the N-Glycan Samples plus LabelingReagent on position#4.
Set the parameters in the Application Settings section:
� Enter the desired labeling reaction duration.
� Enter the desired labeling reaction temperature.
Third section of the orange graphic above.
Default duration is 60 minutes.
Default temperature is 70�C; allowed temperature range is20–70�C.
The temperature may be monitored on the digital readoutof the STC Controller.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the orange graphic above; labware islisted in the second section.
-
29
Click on “Run 5 Labeling.” The Processing Plate containing N-Glycan Samples ismoved from position#4 to position#9, where it is mixedfor one minute. It will remain there until the ThermalStation temperature is within 1�C of the set point (~10minutes to achieve 70�C). Then the plate will be movedto position#4 and the timer starts. When finished, theplate is moved to position#7 and the protocol ends. Theplate movement ensures that the N-Glycan Samples are atthe desired temperature for the set amount of time withno additional temperature ramp up or down.
A status line will show when the protocol is complete: “Run complete. Module idle.”
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
Prepare Cleanup Protocol Reagents during the labeling incubation. Red section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab
NOTE: The maximum volume per channel is 7.0 ml.
• 96% Acetonitrile Solution (CU Priming and Washing Solutions):Add the ultrapure water to a glass, graduated cylinder. Bring the volume up to thecorrect volume with 100% acetonitrile. Transfer to a similarly sized glass storagevessel, cap tightly and swirl gently to mix.
� May be prepared up to one week before use. Storesealed in a similarly sized glass container at roomtemperature.
-
30
• 2-AB Sample Load Solution:
Add the amount of 5x 2-AB Sample Load Solution indicated in the Reagent VolumeCalculator to a glass graduated cylinder using a volumetric pipette. Bring the volumeup to the final volume with HPLC-grade acetonitrile.
Transfer to a similarly sized glass container, cap tightly and swirl gently to mix. Before using, allow solution to come to room temperature and then gently invert tomix contents..
� Prepare on the day of use. Store sealed in a similarlysized, clean, glass container until the Cleanup Protocol inorder to prevent evaporation of the acetonitrile. Precisecontrol of the acetonitrile concentration is important toensure both retention of N-glycans and removal of excessdye during Cleanup.
NOTE: Due to the viscosity and surface tension of the 5x2-AB Sample Load Solution, we recommend using onlyglass cylinders. Use volumetric pipettes to assureaccurate addition of the 5x 2-AB Sample Load Solution;do not use a standard pipette.
It is important to prevent preferential evaporation of theacetonitrile, which affects the performance of the HILICseparation.
At the software prompt, prepare for resuspension of the labeling reaction:
1. Move the plate containing the labeled N-Glycan Samples from position#7 to afume hood.
2. Allow the plate to cool for 5 minutes to room temperature.3. Remove the Aluminum Seal.
Remove the Aluminum Seal in a fume hood to avoidinhalation of hazardous reaction products.
NOTE: Be sure to remove all excess aluminum, as it caninterfere with the AssayMAP Bravo grippers and sensors
Proceed to Protocol 6.
-
31
6 Cleanup Protocol
Remove the CU Cartridge Rack, Receiver Plate and tape from position#6. Replacethe lid to protect any unused Cartridges and store appropriately.
Set the parameters in the Application Settings section: Third section of the red graphic above.
Indicate the Final Eluate Volume. (25 - 100 �l, as needed for the specificanalytical method).
The AssayMAP Bravo elutes the Labeled N-Glycan Samplesquantitatively from the CU Cartridges in 25 �l of water. Any additional elution volume is added in a separate step.
The Labeled N-Glycan Samples are eluted from the CUCartridge as continuous fractions. A 1-minute mixing stepproduces a homogeneous sample, ready for analysis.
-
32
Place the Processing Plate containing the Labeled N-Glycan Samples on position#4.
Place the previously prepared Cleanup Solutions on the deck, making sure that thefilled reservoirs correspond to the CU Cartridge positions:
1. Place the 12-Column, Reservoir Plate containing CU Washing Solution (3 mleach reservoir of 96% Acetonitrile) on position#5.
2. Place the 12-Column, Reservoir Plate containing CU Priming Solution (6 mleach reservoir of 96% Acetonitrile) on position#6.
3. Place a 12-Column, Reservoir Plate containing 2-AB Sample Load Solution onposition#7.
4 Place a 12-Column, Reservoir Plate containing HPLC-grade water on position#8.
After removing the Aluminum Seal in a fume hood, eachwell now contains Labeled N-Glycan Samples plusunreacted dye, which will be removed using HILICchromatography.
Place an empty Square, Deep-Well Plate on position#3 for Waste.
Label an empty PCR Plate “CU Eluate” and place it on position#9.
Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the red graphic above; labware is listed inthe second section.
Click on “Run 6 Cleanup.” The protocol will take approximately 30 minutes.
Status is shown in the status bar.
WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.
-
33
Upon completion of the protocol:
1. Remove the Eluate Collection Plate containing the Labeled N-Glycan Samplesfrom position#9.
2. Proceed directly to analysis, or seal the plate with Aluminum Seal and store at-20�C.
3. Remove all other labware from the deck and discard any remaining solutionsand waste appropriately.
A status line will show when the protocol is complete:“Run complete. Module idle.”
The Labeled N-Glycan Samples are ready for analysis.
NOTE: Waste plate at position#3 contains Acetonitrile;discard according to waste disposal procedures.
Empty the Wash Station waste container.
-
Qu
ickG
uid
e34
QU
ICK
GU
IDE
Gly
co
pro
tein
Sa
mp
le P
late
Pre
pa
ratio
n
Pre
pare
the G
lyco
pro
tein
Samp
le P
late w
ith re
qu
ired
vo
lum
es u
sing th
e Sam
ple
Info
& R
eag
en
t Pre
p tab
in th
e R
eag
en
t Vo
lum
e C
alculato
r to co
mp
ute
starting v
olu
mes.
Disp
en
se th
e G
lyco
pro
tein
Samp
les in
to e
ither a P
CR
Plate
or a U
-Bo
ttom
Plate
dep
en
din
g o
n th
e fin
alD
en
ature
d G
lyco
pro
tein
Samp
le V
olu
me.
NO
TE
: Be su
re n
ot to
intro
du
ce b
ub
ble
s; spin
do
wn
if nece
ssary.
Reag
en
t Sou
rce P
late P
rep
aration
Pre
pare
the re
agen
ts and
solu
tion
s need
ed
as describ
ed
belo
w an
d in
the g
rey sectio
n o
f the R
eag
en
tV
olu
me C
alculato
r.
1.
Dig
estio
n B
uffe
r �
Dig
estio
n B
uffe
r may
be p
rep
ared
up
to o
ne w
eek b
efo
re u
se. Sto
re at 2
–8�C
.
In a se
parate
vial, ad
d th
e am
ou
nts o
f 25x D
igestio
n B
uffe
r and
ultrap
ure
wate
r as ind
icated
in th
eR
eag
en
t Vo
lum
e C
alculato
r, and
then
vo
rtex to
mix
tho
rou
gh
ly.
2.
En
zym
e So
lutio
n
� E
nzy
me So
lutio
n sh
ou
ld b
e p
rep
ared
on
the d
ay o
f use
; store
at RT
.
•V
orte
x th
e v
ial of N
-Gly
canase
to m
ix, an
d th
en
spin
do
wn
brie
fly to
colle
ct the co
nte
nts in
the b
aseo
f the v
ial.
•In
a sep
arate v
ial, add
the am
ou
nts o
f Dig
estio
n B
uffe
r (pre
pare
d ab
ove) an
d N
-Gly
canase
ind
icated
in th
e R
eag
en
t Vo
lum
e C
alculato
r, and
then
vo
rtex to
mix
tho
rou
gh
ly.
3.
Blo
ckin
g R
eag
en
t (p
rovid
ed
with
the K
it)4.
Den
aturatio
n R
eag
en
t (p
rovid
ed
with
the K
it)5.
Fin
ishin
g R
eag
en
t (p
rovid
ed
with
the K
it)
Prin
t the "R
eag
en
t Sou
rce P
late Se
tup
" tab o
f the R
eag
en
t Vo
lum
e C
alculato
r.
Pip
ette
pre
pare
d so
lutio
ns fro
m th
e p
revio
us ste
p in
to e
ach w
ell lo
cation
in th
e R
eag
en
t Sou
rce P
late as
sho
wn
in th
e tab
le in
the "R
eag
en
t Sou
rce P
late Se
tup
" tab.
-
Qu
ickG
uid
e35
Op
tion
al Labelin
g So
urce
Plate
Pre
paratio
n
See Lab
elin
g So
urce
Plate
tab o
f the R
eag
en
t Vo
lum
e C
alculato
r.
No
mo
re th
an o
ne h
ou
r prio
r to u
se, Lab
elin
g R
eag
en
t may
be p
rep
ared
in C
olu
mn
1 o
f the Lab
elin
gSo
urce
Plate
and
transfe
rred
man
ually
by m
ulti-ch
ann
el p
ipette
to th
e N
-gly
can Sam
ple
s in th
eP
roce
ssing P
late.
Pre
paratio
n o
f the A
ssayM
AP
Brav
o an
d A
ssociate
d E
qu
ipm
en
t
Tu
rn o
n th
e A
ssayM
AP
Brav
o, co
mp
ute
r, pu
mp
and
Inh
eco
con
trolle
r.
Op
en
the A
ssayM
AP
Laun
chP
ad w
eb
inte
rface fro
m th
e d
esk
top
to acce
ss the R
eag
en
t Vo
lum
eC
alculato
r, Gly
ko
Pre
p-p
lus P
roto
cols an
d su
pp
ort d
ocu
men
ts.
Un
der th
e N
-Gly
can Sam
ple
Pre
p tab
, click o
n th
e 2
-AB
icon
to lau
nch
N-G
lycan
Samp
le P
rep
: RX
dig
estio
n &
2-A
B lab
elin
g.
Verify
that th
e w
ash statio
n is in
stalled
on
po
sition
#1.
•C
on
nect ap
pro
priate
tub
ing fro
m D
I wate
r con
tainer to
the p
um
p, an
d fro
m th
e p
um
p to
the w
ashstatio
n, an
d fro
m th
e w
ash statio
n to
the w
aste co
ntain
er.
•Fill th
e w
ash statio
n so
urce
con
tainer w
ith D
I wate
r (if nece
ssary).
•E
mp
ty th
e w
ash statio
n w
aste co
ntain
er (if n
ece
ssary).
•C
lick o
n "P
rime an
d W
ash" to
prim
e th
e ch
imn
eys.
•V
erify
that all ch
imn
eys h
ave w
ater flo
win
g.
Verify
that th
e P
eltie
r Th
erm
al Station
with
PC
R P
late A
dap
ter is in
stalled
on
po
stion
#4.
Verify
that th
e O
rbital Sh
akin
g Statio
n is in
stalled
on
po
sition
#9.
WA
RN
ING
: Do
no
t ever re
ach in
to th
e A
ssayM
AP
Brav
o sp
ace w
hile
it is op
eratin
g. T
his w
ill bre
ak th
ese
curity
line; th
e A
ssayM
AP
Brav
o w
ill mak
e an
em
erg
en
cy sto
p, an
d m
ay n
ot b
e ab
le to
be re
started
with
ou
t inte
rven
tion
from
a kn
ow
led
geab
le o
perato
r.
-
Qu
ickG
uid
e36
1P
late
& R
ea
ge
nt S
etu
p P
roto
co
l
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
:
1.
Nu
mb
er o
f Co
lum
ns to
Man
age (co
lum
ns o
f 8 sam
ple
s) = _
________
2.
Starting C
olu
mn
of D
estin
ation
Plate
= _
________
Ch
eck
the p
revio
usly
pre
pared
Reag
en
t Sou
rce P
late fo
r bu
bb
les; ce
ntrifu
ge b
riefly
to cle
ar bu
bb
les if n
ece
ssary.
Place
the R
eag
en
t Sou
rce P
late o
n p
ositio
n#5.
Place
the e
mp
ty Se
ating Statio
n o
n p
ositio
n#3.
Label a cle
an P
CR
Plate
as "Pro
cessin
g P
late" an
d p
lace it o
n th
e H
eate
r Station
Ad
apto
r on
po
sition
#4
.
Place
a Pip
ette
-Tip
Bo
x o
n p
ositio
n#6; R
EM
OV
E T
HE
LID.
Label an
d stack
4 U
-Bo
ttom
Plate
s:a.
Dig
estio
n B
uffe
r (top
)b
.B
lock
ing R
eag
en
tc.
Den
aturatio
n R
eag
en
td
.Fin
ishin
g R
eag
en
t (bo
ttom
)
Place
the stack
on
po
sition
#7.
Click
"Ru
n P
roto
col 1
"
Th
e ap
pro
xim
ate ru
n tim
e is 1
5 m
inu
tes. A
Status lin
e w
ill sho
w w
hen
the p
roto
col is co
mp
lete
: "Ru
nco
mp
lete
. Mo
du
le id
le."
Pro
ceed
to P
roto
col 2
.
-
Qu
ickG
uid
e37
2R
X C
artrid
ge
Se
tup
Pro
toc
ol
Rearran
ge th
e d
eck
in p
rep
aration
for p
roce
ssing:
3.
Rem
ove th
e R
eag
en
t Sou
rce P
late fro
m p
ositio
n#2; d
iscard.
4.
Rem
ove th
e Se
ating Statio
n o
n p
ositio
n#3 an
d d
iscard th
e u
sed
pip
ette
tips.
5.
Place
the n
ow
em
pty
Seatin
g Statio
n o
n p
ositio
n#2.
6.
Rem
ove th
e lid
from
a Rack
of R
X C
artridges an
d p
lace it w
ith its R
ece
iver P
late o
n p
ositio
n#6.
T
ape th
e R
X C
artridge R
ack an
d R
ece
iver P
late to
the d
eck
.
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
:
1.
Ind
icate th
e first co
lum
n o
f RX
Cartrid
ges to
be u
sed
in th
e R
X C
artridge R
ack in
po
sition
#6.
2.
Ind
icate th
e n
um
ber o
f colu
mn
s to tran
sfer. T
he co
lum
ns d
irectly
follo
w th
e first co
lum
nin
dicate
d ab
ove in
the le
ft to rig
ht d
irectio
n.
3.
Ind
icate th
e first co
lum
n in
the Se
ating Statio
n in
to w
hich
these
colu
mn
s of R
X C
artridges w
ill be
place
d.
Mak
e su
re th
at the A
ssayM
AP
Brav
o d
eck
loo
ks lik
e th
e D
eck
Layo
ut sh
ow
n ab
ove.
Click
on
"Ru
n 2
RX
Cartrid
ge Se
tup
."
Th
e ap
pro
xim
ate ru
n tim
e is 1
min
ute
. A Statu
s line w
ill sho
w w
hen
the p
roto
col is co
mp
lete
: “Ru
nco
mp
lete
. Mo
du
le id
le.”
Pro
ceed
to P
roto
col 3
.
-
Qu
ickG
uid
e38
3Im
mo
biliz
atio
n &
Dig
es
tion
Pro
toc
ol
Disp
en
se th
e R
X P
rimin
g So
lutio
n (1
00 %
Ace
ton
itrile) in
to a 1
2-C
olu
mn
Rese
rvo
ir Plate
, corre
spo
nd
ing
to th
e co
lum
ns o
f RX
Cartrid
ges to
be p
roce
ssed
.
Fill th
e ch
ann
els w
ith th
e v
olu
me sp
ecifie
d in
the B
lue se
ction
of th
e p
rinted
Reag
ent V
olu
me C
alculato
r“Sam
ple In
fo &
Reag
ent P
rep
” tab.
Rearran
ge th
e d
eck
in p
rep
aration
for p
roce
ssing:
1.
Rem
ove th
e R
X C
artridge R
ack, R
ece
iver P
late an
d tap
e fro
m p
ositio
n#6. R
ep
lace th
e lid
top
rote
ct any re
main
ing R
X C
artridges an
d sto
re ap
pro
priate
ly.
2.
Place
the 1
2-C
olu
mn
, Rese
rvo
ir Plate
con
tainin
g th
e R
X P
rimin
g So
lutio
n o
n p
ositio
n#3, m
akin
gsu
re th
at the fille
d re
serv
oirs co
rresp
on
d to
the R
X C
artridge p
ositio
ns.
3.
Place
the G
lyco
pro
tein
Samp
le P
late o
n p
ositio
n#6.
Set th
e sam
ple
plate
typ
e in
the “Lab
ware
” sectio
n, lin
e 6
abo
ve:
Sele
ct PC
R P
late (lo
w v
olu
me) o
r U-B
otto
m P
late (h
igh
vo
lum
e).
-
Qu
ickG
uid
e39
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
(Th
ird se
ction
of th
e b
lue g
raph
ic abo
ve.):
1.
En
ter th
e D
en
aturan
t Vo
lum
e (ce
ll D16 in
the R
eag
en
t Vo
lum
e C
alculato
r).
2.
En
ter th
e Startin
g Sam
ple
Vo
lum
e (ce
ll D15 in
the R
eag
en
t Vo
lum
e C
alculato
r).
3.
Den
ature
d Sam
ple
Vo
lum
e (calcu
lated
valu
e, g
rey).
4.
En
ter th
e Sam
ple
Load
ing F
low
Rate
.
5.
En
ter th
e T
em
peratu
re Se
t Po
int fo
r Dig
estio
n.
6.
En
ter th
e D
uratio
n o
f the D
igestio
n ste
p.
Mak
e su
re th
at the A
ssayM
AP
Brav
o d
eck
loo
ks lik
e th
e D
eck
Layo
ut ab
ove.
Click
on
“Ru
n 3
Imm
ob
ilization
& D
igestio
n.”
Th
e p
roto
col w
ill take b
etw
een
90 an
d 1
20 m
inu
tes d
ep
en
din
g o
n th
e sam
ple
vo
lum
e an
d in
cub
ation
time. T
he statu
s line w
ill sho
w w
hen
the p
roto
col is co
mp
lete
: "Ru
n co
mp
lete
. Mo
du
le id
le."
Th
e P
roce
ssing P
late (p
ositio
n#4) n
ow
con
tains th
e N
-gly
cans; re
mo
ve fo
r dry
ing. T
he A
ssayM
AP
Brav
om
ay b
e sh
utd
ow
n at th
is po
int.
Pro
ceed
to M
anu
al Pro
cessin
g.
Ma
nu
al P
roc
es
sin
g - D
ryin
g th
e N
-Gly
ca
ns p
rior to
lab
elin
g
Rearran
ge th
e d
eck
in p
rep
aration
for p
roce
ssing:
•R
em
ove th
e P
roce
ssing P
late (P
CR
plate
) from
po
sition
#4.
•D
ry th
e N
-Gly
can Sam
ple
s usin
g a ce
ntrifu
gal e
vap
orato
r with
the h
eat se
tting tu
rned
off u
ntil fu
llyd
ry (2
0 m
inu
tes to
1 h
ou
r).
Pro
toco
l 4 m
ay b
e p
roce
ssed
simu
ltaneo
usly
with
Man
ual P
roce
ssing.
-
Qu
ickG
uid
e40
4C
U C
artrid
ge
Se
tup
Pro
toc
ol
Rearran
ge th
e d
eck
in p
rep
aration
for p
roce
ssing:
•R
em
ove all Im
mo
bilizatio
n an
d D
igestio
n lab
ware
from
deck
.•
Discard
use
d R
X C
artridges.
Rem
ove th
e lid
from
a Rack
of C
U C
artridges an
d p
lace it w
ith its R
ece
iver P
late o
n p
ositio
n#6.
Tap
e th
e C
U C
artridge R
ack an
d R
ece
iver P
late to
the d
eck
.
Place
the e
mp
ty Se
ating Statio
n in
po
sition
#2.
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
:
1. In
dicate
the first co
lum
n fro
m w
hich
to tak
e th
e C
U C
artridges fro
m R
ack in
po
sition
#6.
2. In
dicate
the n
um
ber o
f colu
mn
s to tran
sfer.
3. In
dicate
the first co
lum
n in
the Se
ating Statio
n w
here
the C
U C
artridges are
to b
e p
laced
.
Mak
e su
re th
at the A
ssayM
AP
Brav
o d
eck
loo
ks lik
e th
e D
eck
Layo
ut sh
ow
n ab
ove.
Click
on
"Ru
n 4
CU
Cartrid
ge Se
tup
."
Th
e ap
pro
xim
ate ru
n tim
e is 1
min
ute
. Wh
en
the N
-gly
can Sam
ple
s are d
ry, p
roce
ed
to M
anu
alP
roce
ssing - Lab
elin
g th
e N
-Gly
cans w
ith 2
-AB
.
-
Qu
ickG
uid
e41
Ma
nu
al P
roc
es
sin
g - L
ab
elin
g th
e N
-Gly
ca
ns w
ith 2
-AB
Pre
pare
Labelin
g R
eagen
t (oran
ge se
ction
of th
e p
rinted
Reag
ent V
olu
me C
alculato
r “Samp
le Info
& R
eag
ent
Pre
p” tab
).
� P
rep
are n
o m
ore
than
on
e h
ou
r befo
re u
se
Allo
w th
e 2
-AB
Solu
tion
and
Red
uctan
t Solu
tion
vials to
com
e to
roo
m te
mp
eratu
re in
the se
aled
desiccan
t bag
befo
re re
mo
vin
g th
em
. Invert e
ach to
mix
gen
tly.
NO
TE
: 2-A
B Lab
elin
g R
eag
en
t com
po
nen
ts are h
azardo
us. P
lease
refe
r to th
e Safe
ty D
ata Sheets o
n o
ur
web
site. P
erfo
rm th
is pro
ced
ure
usin
g ap
pro
priate
perso
nal safe
ty p
rote
ction
, eyeglasse
s and
nitrile
glo
ves.
In a se
parate
vial, ad
d th
e am
ou
nts o
f 2-A
B So
lutio
n an
d R
ed
uctan
t Solu
tion
as ind
icated
in th
eR
eag
en
t Vo
lum
e C
alculato
r, and
then
vo
rtex to
mix
tho
rou
gh
ly.
Op
tion
al: Pip
ette
eq
ual v
olu
mes o
f 2-A
B Lab
elin
g R
eag
en
t into
each
well o
f the first co
lum
n o
f the
Labelin
g So
urce
Plate
(PC
R p
late) as in
structe
d in
the Lab
elin
g So
urce
Plate
tab o
f the R
eag
en
t Vo
lum
eC
alculato
r.
Ad
d th
e Lab
elin
g R
eag
en
t to d
ired
N-g
lycan
s in th
e P
roce
ssing P
late:
�A
dd
5 μ
L of Lab
elin
g R
eag
en
t to th
e sid
e o
f each
N-G
lycan
Samp
le w
ell ab
ou
t half w
ay d
ow
n th
esid
e o
f the w
ell.
�T
ap th
e p
late sh
arply
on
the b
en
ch to
en
sure
that th
e d
rop
let o
f Labelin
g R
eag
en
t dro
ps to
the
bo
ttom
of th
e w
ell.
�C
over th
e p
late w
ith th
e A
lum
inu
m Se
al pro
vid
ed
.
Pro
ceed
to P
roto
col 5
.
-
Qu
ickG
uid
e42
5L
ab
elin
g P
roto
co
l
Place
the se
aled
Pro
cessin
g P
late co
ntain
ing th
e N
-Gly
can Sam
ple
s plu
s Labelin
g R
eag
en
t on
po
sition
#4.
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
abo
ve:
•E
nte
r the d
esire
d lab
elin
g re
action
du
ration
. Th
e d
efau
lt du
ration
is 60 m
inu
tes.
•E
nte
r the d
esire
d lab
elin
g re
action
tem
peratu
re. T
he d
efau
lt tem
peratu
re is 7
0�C
; allow
ed
tem
peratu
reran
ge is 2
0 - 70�C
.
Mak
e su
re th
at the A
ssayM
AP
Brav
o d
eck
loo
ks lik
e th
e D
eck
Layo
ut sh
ow
n ab
ove.
Click
on
"Ru
n 5
Labelin
g.”
Du
ring th
e lab
elin
g in
cub
ation
, pre
pare
the C
lean
up
Pro
toco
l reag
en
ts as ind
icated
in th
e R
ed
sectio
n o
fth
e p
rinte
d R
eag
en
t Vo
lum
e C
alculato
r “Samp
le In
fo &
Reag
en
t Pre
p” tab
:
•96%
Ace
ton
itrile So
lutio
n (C
U P
rimin
g an
d W
ashin
g So
lutio
ns):
� M
ay b
e p
rep
ared
up
to o
ne w
eek b
efo
re u
se. Sto
re se
aled
in a sim
ilarly size
d g
lass con
tainer at ro
om
tem
peratu
re.
Ad
d th
e u
ltrapu
re w
ater to
a glass, g
radu
ated
cylin
der.
Brin
g th
e v
olu
me u
p to
the co
rrect v
olu
me w
ith 1
00%
aceto
nitrile
.
Tran
sfer to
a similarly
sized
, glass sto
rage v
esse
l.
Cap
tigh
tly an
d sw
irl gen
tly to
mix
.
-
Qu
ickG
uid
e43
•2-A
B Sam
ple
Load
Solu
tion
:
� P
rep
are o
n th
e d
ay o
f use
. Store
seale
d in
a similarly
sized
, clean
, glass co
ntain
er u
ntil th
e C
lean
up
pro
toco
l.
Ad
d th
e am
ou
nt o
f 5x 2
-AB
Samp
le Lo
ad So
lutio
n in
dicate
d in
the R
eag
en
t Vo
lum
e C
alculato
r to a
glass g
radu
ated
cylin
der u
sing a v
olu
metric p
ipette
.
Brin
g th
e v
olu
me u
p to
the fin
al vo
lum
e w
ith H
PLC
-grad
e ace
ton
itrile.
Tran
sfer to
a similarly
sized
glass co
ntain
er, cap
tigh
tly an
d sw
irl gen
tly to
mix
.
Allo
w so
lutio
n to
com
e to
roo
m te
mp
eratu
re an
d th
en
gen
tly in
vert to
mix
con
ten
ts befo
re u
sing.
At th
e so
ftware
pro
mp
t, pre
pare
for re
susp
en
sion
of th
e lab
elin
g re
action
:
1.
Mo
ve th
e p
late co
ntain
ing th
e lab
ele
d N
-Gly
can Sam
ple
s from
po
sition
#7 to
a fum
e h
oo
d.
2.
Allo
w th
e p
late to
coo
l for 5
min
ute
s to ro
om
tem
peratu
re.
3.
Rem
ove th
e A
lum
inu
m Se
al.
NO
TE
: Rem
ove th
e A
lum
inu
m Se
al in a fu
me h
oo
d to
avo
id in
halatio
n o
f hazard
ou
s reactio
n p
rod
ucts.
Pro
ceed
to P
roto
col 6
.
-
Qu
ickG
uid
e44
6C
lea
nu
p P
roto
co
l
Rem
ove th
e C
U C
artridge R
ack, R
ece
iver P
late an
d tap
e fro
m p
ositio
n#6. R
ep
lace th
e lid
and
store
app
rop
riately
.
Set th
e p
aramete
rs in th
e A
pp
lication
Settin
gs se
ction
abo
ve:
Ind
icate th
e F
inal E
luate
Vo
lum
e (2
5-1
00 �
l).
Afte
r rem
ovin
g th
e A
lum
inu
m Se
al in a fu
me h
oo
d, p
lace th
e P
roce
ssing P
late co
ntain
ing th
e Lab
ele
dN
-Gly
can Sam
ple
s on
po
sition
#4.
Place
the p
revio
usly
pre
pare
d C
lean
up
Solu
tion
s on
the d
eck
, mak
ing su
re th
at the fille
d re
serv
oirs
corre
spo
nd
to th
e C
U C
artridge p
ositio
ns:
•P
lace th
e 1
2-C
olu
mn
, Rese
rvo
ir Plate
con
tainin
g C
U W
ashin
g So
lutio
n (3
ml e
ach re
serv
oir o
f 96%
Ace
ton
itrile) o
n p
ositio
n#5.
•P
lace th
e 1
2-C
olu
mn
, Rese
rvo
ir Plate
con
tainin
g C
U P
rimin
g So
lutio
n (6
ml e
ach re
serv
oir o
f 96%
Ace
ton
itrile) o
n p
ositio
n#6.
•P
lace a 1
2-C
olu
mn
, Rese
rvo
ir Plate
con
tainin
g 2
-AB
Samp
le Lo
ad So
lutio
n o
n p
ositio
n#7.
•P
lace a 1
2-C
olu
mn
, Rese
rvo
ir Plate
con
tainin
g H
PLC
-grad
e w
ater o
n p
ositio
n#8.
Place
an e
mp
ty, Sq
uare
, Deep
-Well P
late o
n p
ositio
n#3 fo
r Waste
.
Place
an e
mp
ty P
CR
Plate
on
po
sition
#9 fo
r Elu
ate.
-
Qu
ickG
uid
e45
Mak
e su
re th
at the A
ssayM
AP
Brav
o d
eck
loo
ks lik
e th
e D
eck
Layo
ut sh
ow
n ab
ove.
Click
on
"Ru
n 6
Cle
anu
p."
Up
on
com
ple
tion
of th
e p
roto
col:
a.R
em
ove th
e E
luate
Co
llectio
n P
late co
ntain
ing th
e Lab
ele
d N
-Gly
can Sam
ple
s from
po
sition
#9.
Th
e Lab
ele
d N
-Gly
can Sam
ple
s are re
ady fo
r analy
sis.
b.
Pro
ceed
dire
ctly to
analy
sis, or se
al the p
late w
ith A
lum
inu
m Se
al and
store
at -20�C
.
c.R
em
ove all o
ther lab
ware
from
the d
eck
and
discard
any re
main
ing so
lutio
ns an
d w
asteap
pro
priate
ly.
NO
TE
: Waste
plate
at po
sition
#3 co
ntain
s Ace
ton
itrile; d
iscard acco
rdin
g to
waste
disp
osal p
roce
du
res.
Em
pty
the W
ash Statio
n w
aste co
ntain
er.
If the n
ext p
roce
ssing is N
OT
a Gly
ko
Pre
p w
ork
flow
, rem
ove all ap
plian
ces fro
m th
e A
ssayM
AP
Brav
od
eck
.
NO
TE
: Th
e p
rese
nce
of th
e W
ash Statio
n, th
e T
herm
al Station
and
the O
rbital Sh
aker can
cause
dam
age
to th
e A
ssayM
AP
Brav
o H
ead
if the n
ext w
ork
flow
has n
ot b
een
defin
ed
to in
clud
e th
em
.
-
46
AN
AL
YS
IS O
F L
AB
EL
ED
N-G
LY
CA
NS
Use stan
dard
tech
niq
ues, su
ch as Liq
uid
Ch
rom
atograp
hy (LC
), Mass Sp
ectro
metry
(MS), o
r a com
bin
ation
of th
etw
o, to
analy
ze th
e aq
ueo
us e
luate
con
tainin
g elu
ted
, labele
d N
-gly
cans (se
e T
ips an
d H
ints, b
elo
w).
TIP
S &
HIN
TS
Op
timizin
g E
xcitatio
n/E
missio
n W
avele
ngth
s
Op
timal e
xcitatio
n/e
missio
n w
avelen
gth
s for 2
-AB
Dye co
nju
gated
to an
N-g
lycan
may
vary
dep
en
din
g u
po
n th
eo
ptical co
nfig
uratio
n o
f the in
strum
en
t use
d. E
xcitatio
n/e
missio
n p
airs that h
ave b
een
use
d to
geth
er in
clud
e250/4
28 n
m (M
elm
er et al., 20
10), 33
0/42
0 n
m (B
igge e
t al., 199
5) and
360
/428
nm
(use
d b
y Pro
Zym
e w
ith th
eW
aters A
cqu
ity U
PLC
; Hax
o e
t al., 2012).
®®
®
Reco
very
of th
e D
egly
cosy
lated
Pro
tein
from
the D
igestio
n (R
X) C
artridge
Ofte
n, th
e d
egly
cosy
lated
pro
tein
is analy
zed
to e
valu
ate th
e co
mp
lete
ness o
f degly
cosy
lation
usin
g su
chele
ctrop
ho
retic m
eth
od
s as SDS-P
AG
E o
r micro
fluid
ic lab-o
n-a-ch
ip te
chn
olo
gy. P
lease co
ntact u
s for g
uid
elin
es
for elu
ting yo
ur g
lyco
pro
tein
from
the R
X C
artridge.
Calcu
lating th
e M
ass of G
lycan
s Labele
d w
ith 2
-AB
Th
e re
du
ctive am
inatio
n re
action
resu
lts in th
e lo
ss of an
oxygen
atom
. Th
e m
ass of th
e 2
-AB
-labele
d N
-gly
can is o
btain
ed
usin
g th
e fo
llow
ing fo
rmu
la:
Gly
can
2-A
B
2-A
B-L
ab
ele
d G
lyca
nO
xygen
Mass
+ M
ass=
Mass
- Mass
Mass A
dd
ed
to G
lycan
Mo
no
isoto
pic
120.0
6875
Averag
e120.2
Dire
ct LC A
naly
sis of N
-Gly
cans A
fter E
lutio
n
If N-G
lycan
Samp
les w
ill be an
alyze
d b
y LC
directly
follo
win
g e
lutio
n fro
m th
e C
U C
artridges, co
llectio
n p
latesm
ay b
e h
eat se
aled
with
pie
rceable
foil (e
.g. T
herm
o E
asy P
ierce 2
0um
Fo
il, #AB
-1720) u
sing a m
icrop
late heat
seale
r (e.g
. Th
erm
o A
LPS 5
0 V
Sem
i auto
mated
Micro
plate
Heat Se
aler, #A
B-1
443).
-
47
RE
FE
RE
NC
ES
Big
ge, J. C
., T. P
. Pate
l, J. A. B
ruce, P
. N. G
ou
ldin
g, S
. M. C
harle
s, R. B
. Pare
kh
. No
nse
lectiv
e a
nd
Effic
ien
t Flu
ore
scen
t Lab
elin
g o
f Gly
can
sU
sing
2-A
min
o B
en
zam
ide a
nd
An
thra
nilic
Acid
. An
al B
ioch
em
23
0: 2
29–38
(1995
).
Me
lme
r, M., T
. Sta
ng
ler, M
. Sch
iefe
rme
ier, W
. Bru
nn
er, H
. To
ll, A. R
up
pre
ch
ter, W
. Lin
dn
er a
nd
A. P
rem
stalle
r. HIL
IC A
naly
sis of
Flu
ore
scen
ce-L
ab
ele
d N
-Gly
can
s from
Reco
mb
inan
t Bio
ph
arm
aceu
ticals. A
na
l Bio
an
al C
he
m 3
98
: 905–91
4 (2
010).
Hax
o, T
., J. Hy
ch
e, M
. Kim
zey
, S. L
ock
hart, C
. Nish
ida, S
. Po
urk
av
eh
, J. We
gste
in, Y
. Q. Y
u a
nd
D. J. P
hillip
s. An
Inte
gra
ted
Stra
teg
y fo
rN
-Gly
can
Sam
ple
Pre
para
tion
an
d A
naly
sis Su
itab
le fo
r All S
tag
es o
f Th
era
pe
utic
Pro
tein
Disc
ov
ery
, Ch
ara
cte
rizatio
n, M
an
ufa
ctu
re a
nd
Qu
ality
Re
lease
. Po
ster se
ssion
pre
sen
ted
at: W
ell C
hara
cte
rized
Bio
tech
no
log
y P
harm
ace
utic
als 1
6 S