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.


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.



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


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”.


Approximate run time is 1 minute.


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.


� 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.



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.


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

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QU

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GU

IDE

Gly

co

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mp

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late w

ith re

qu

ired

vo

lum

es u

sing th

e Sam

ple

Info

& R

eag

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t Pre

p tab

in th

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lum

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r to co

mp

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mes.

Disp

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se th

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tein

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re n

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ce b

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ssary.

Reag

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aration

Pre

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the re

agen

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solu

tion

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ed

as describ

ed

belo

w an

d in

the g

rey sectio

n o

f the R

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en

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r.

1.

Dig

estio

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Dig

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rep

ared

up

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ne w

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re u

se. Sto

re at 2

–8�C

.

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parate

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d th

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uffe

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ultrap

ure

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icated

in th

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� E

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ld b

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ay o

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.

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spin

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in th

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it)

Prin

t the "R

eag

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t Sou

rce P

late Se

tup

" tab o

f the R

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Qu

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Op

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ually

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-AB

icon

to lau

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dig

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2-A

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n is in

stalled

on

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ssary).

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prim

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Verify

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with

PC

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n P

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ceed

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.

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Rem

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6.

Rem

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from

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of R

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ith its R

ece

iver P

late o

n p

ositio

n#6.

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ape th

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artridge R

ack an

d R

ece

iver P

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the d

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.

Set th

e p

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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

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d ab

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ft to rig

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irectio

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3.

Ind

icate th

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n in

the Se

ating Statio

n in

to w

hich

these

colu

mn

s of R

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artridges w

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place

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Mak

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re th

at the A

ssayM

AP

Brav

o d

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loo

ks lik

e th

e D

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Layo

ut sh

ow

n ab

ove.

Click

on

"Ru

n 2

RX

Cartrid

ge Se

tup

."

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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

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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

GlykoPrep - plus Rapid N-Glycan Sample Preparation with 2 ......Labware Set from ProZyme (product...

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