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N-Glycan Purification & Labelling Kits

O-Glycan Sample Preparation Kit

Glycan Array

GLYCAN ANALYSISPRODUCTS AND SERVICES

EZGlyco™ mAb-N Kit with 2-ABStreamlined and rapid N-glycan preparation from IgG

HPLC-ready glycan preparation in less than 2 hours Purify IgG from cell culture supernatant

Release and label N-glycans

o Additional required reagents• Milli-Q grade® water• Acetonitrile• Acetic acid• Dimethyl sulfoxide

o Additional required consumables• 2.0-mL microcentrifuge tubes• 1.5-mL microcentrifuge tubes

o Additional required equipment• Centrifuge for sample tubes• Heat block

10 µg hIgG was spiked in culture mediumand 2-AB-labeled N-glycans were preparedaccording to the instructions. An aliquot of0.2 µg IgG was analyzed using HPLC.

• Generates equivalent glycan profiles to conventional methods• Reproducible peak area (% of total) was observed with CV values typically less than 10%.

13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 min

0.00

0.25

0.50

0.75

1.00

mV(x10)検出器A:Ex:330nm,Em:420nm

Retention Time (min)

1

2

3

4

5

6

1: Antibody Capturing Column (Qty 10)2: Antibody Capturing Solution (Qty 1)3: Washing Buffer (Qty 1)4: Glycan Release Enhancer (Qty 1)5: PNGase F Solution (Qty 1)6: 2-Aminobenzamide (Qty 1)7: Reducing Reagent (Qty 1)8: Cleanup Column (Qty 10)9: Instruction (Qty 1)

Catalog No.: BS-X4410ZItem: EZGlyco™ mAb-N Kit with 2-ABQuantity: 1 kit for 10 assaysStore at 4°C

BRINGING GLYCOMICS TO LIFEⓇ

The EZGlyco™ mAb-N Kit with 2-AB revolutionary streamlines N-glycan sample preparation from IgG in crudematerials, such as cell culture supernatant and biological fluids, by accelerated deglycosylation and fluorescent labelingreactions in a single column. The Kit is developed to circumvent multiple sample transfers and sample drying steps andbypass a separate IgG purification step thus making it simplified and fast in operation. The Kit includes enzyme,reagents, and accessories necessary for completion of HPLC-ready N-glycan preparation in less than 2 hours ofhandling time. It provides traditional 2-aminobenzamide labeling designed with a non-toxic reductant.

Sample preparation workflow withEZGlyco mAb-N Kit with 2-AB

Contents

Ordering Information

Application Data

N-Glycan profiling of human IgG using the EZGlyco™mAb-N Kit with 2-AB.

BlotGlyco® -Glycan Purification and Labeling Kit-

1. BlotGlyco® beads and labeling reagent2. Reaction tube3. Cleanup column for eliminating excess reagent4. Sample tube5. Protocol and application notes

S

HzPolymer beads are densely coated with immobilized hydrazide groups to facilitate glycan capture

HZ:-NHNH2BlotGlyco® beads1

2 34

5

Kit Components

49 kinds of N-glycans

were detected.

Application

Removal of proteins and peptides to provide cleaner MALDI MS scans

Screening glycans from fermentation broth to find optimum harvest time

Clone Screening in order select strains which produce high activity glycoforms

Screening N- and O-glycans in human sera

Other applications

Hz

BlotGlyco® beads ★：Labeling compound

Glycan

Principle

Biological sample

1: Capturing of glycans 2: Releasing and labeling 3: Cleanup

1 2 3MSHPLC

BlotGlyco® including hydrazide beads and theirapplications are covered by various Japan and international patents. The product specifications are subject to change without notice.

1500 1750 2000 2250 2500 2750 3000 3750m/z

3250 35001250

Ex. N-glycnas of human serum: using 5 µL serum

GlcNAc

Man

Fuc

Gal

NeuGc

NeuAc

Cat. No Product Application BlotGlyco® BeadsLabeling reagent

for MALDI-TOF MS

Reaction spin

tubeCleanup Column

BS-45403Z BlotGlyco® 10 MALDI-TOF MS For 10 assays For 10 assays 10 10

BS-45614Z BlotGlyco® 10B HPLC For 10 assays - 10 10




BS-45406Z BlotGlyco® 100A MALDI-TOF MS For 100 assays For 100 assays - -


BS-45408Z BlotGlyco® 100C HPLC For 100 assays - - -

BS-45409Z BlotGlyco® 100D HPLC For 100 assays - - 100

BS-45410Z BlotGlyco® 200C HPLC For 200 assays - - -

BS-45411Z BlotGlyco® 100E HPLC For 100 assays - 100 -

BS-45412Z BlotGlyco® 200E HPLC For 200 assays - 200 -

BS-45413Z BlotGlyco® 96-plate B HPLC For 96 assays - 96-well fi lter plate 96-well cleanup plate

Heating block for multiwell plateVacuum manifold

96-well filter plate

Dispense BlotGlyco® beads and sample into each well of the filter plate. Instead of centrifugation, vacuum aspiration is usable. Glycan purification, labeling and removal of excess labeling reagents are completed in 96-well plate format.

BlotGlyco® -Glycan Purification and Labeling Kit-96-well filter plate format

Useful for screening, process development and quality control of biopharmaceuticals Suitable for biormarker discovery from crude biological samples

BlotGlyco® O-Glycan Sample Preparation Kit

[Key features] BlotGlyco® O-Glycan Sample Preparation Kit eliminates time-consuming tedious purification steps. A two-day procedure

results in highly purified, 2AB labeled O-glycan sample preparation. Use of a mild ammonium salt for the releasing reaction, and as a consequence, the operations are much safe and simple over

hydrazinolysis. Easy operations. No need for special devices.

[BlotGlyco® O-Glycan Sample Preparation Kit Components]• O-Glycan release reagent (for 10 tests)• BlotGlyco® Beads (for 10 tests)• Reaction spin tubes(for 10 tests)• Sample tubes(for 10 tests)• Cleanup columns(for 10 tests)• Operation Protocol

Figure 1. Process of O-glycan sample preparation from glycoprotein using BlotGlyco® O-Glycan Sample Preparation Kit

“BlotGlyco® O-Glycan Sample Preparation Kit” enables scientists to analyze O-linked glycans attaching to glycoproteins in biological samples, including biotherapeutic glycoproteins, in a simple, safe and streamlined operation. BlotGlyco® O-Glycan Sample Preparation Kit is a valuable tool to support customers involved in glycoprotein characterization in research and process development, monitoring and batch release, characterization of previously unattainable information on O-glycan structures.

BlotGlyco® O-Glycan Sample Preparation Kit consists of “O-glycan releasing reagent” and “BlotGlyco® bead”. “O-Glycan releasing reagent” is for chemical treatment of protein samples with an ammonium salt that is appreciably innocuous than conventional methods. Besides, BlotGlyco® O-Glycan Sample Preparation Kit provides an effective O-glycan preparation with unbiased mild O-glycan release from glycoproteins. Since the released O-glycans from proteins carry a reducing terminal that is available for further manipulations, O-glycans can be enriched and labeled using BlotGlyco® bead included in the BlotGlyco® O-Glycan Sample Preparation Kit. Even smaller O-glycans such as mono-, di-, and trisaccharides can be recovered efficiently owing to chemoselective ligation between their reducing aldehyde and hydrazide group on the beads. Thus, the BlotGlyco® O-Glycan Sample Preparation Kit is beneficial for a wide range of users who wish to recover O-glycans with a specific (fluorescent) tag depending upon user’s analysis method, e.g. HPLC/UPLC, LC-MS, and capillary electrophoresis, etc.

Step 1. Release O-glycans from glycoproteins with a mild ammonium salt (5 hours reaction plus an overnight dry-up)Step 2. Purify and label the released O-glycans with BlotGlyco® bead (1 day)

Glycoprotein

N-glycanO-glycan

AspSer/Thr

BlotGlyco® bead

LCMS Asp

Ser/Thr

[Comparability with a conventional method]

-2AB-2AB -2AB

-2AB

Hydrazinolysismethod

Flu

ore

scen

ceFl

uo

resc

ence

BlotGlyco®O-Glycan Sample Preparation Kit

Figure 4. Reproducibility of BlotGlyco® O-Glycan Sample Preparation Kit

Figure 2. HPLC analysis of bovine fetuin (20 µg) O-glycan using BlotGlyco® O-Glycan Sample Preparation Kit and a hydrazinolysis method.

Figure 3. Comparison of peeling product with hydrazinolysis method

BlotGlyco® O-Glycan Sample Preparation Kit

Hydrazinolysis method

Peeling product

Rel

ativ

e p

eak

area

(%

)

N=12

Cat. No. Product name O-Glycan releasing reagent BlotGlyco® bead Reaction tube Cleanup column

BS-45450Z BlotGlyco® O -Glycan Sample Preparation Kit for 10 tests for 10 tests for 10 tests for 10 tests

[Ordering information]

Glycan Array-For multiple analysis of glycan-related interaction-

GlycanArrays are useful in studies such as:

• Interaction of microorganism

• Analysis of glycan-binding proteins

• Autoantibody, etc.

ApplicationsComponents

Cover slideGlycanArrayProtocol

GlycanArray

Cover slide

glycanPlastic slide

Proteins such as lectins, antibodies, toxins, etc.

Pathogens such as viruses,bacteria, microorganisms, etc.

Example of detection

High S/N ratio

Glycan structures (28 types)

・N-glycan・・・M9, NA2, A2, NA2F, NA3, A3, NA4・O-glycan・・・STn, T・GAG ・・・ Heparin, Desulfated heparin(4 types)・Lewis series ・・・Lea, Lex, Slea, SLex

・Lac series ・・・Lac, a2-3Sialyl Lac, a2-6Sialyl Laca2-3Sialyl LacNAc, a2-6Sialyl LacNAcLacto-N-tetraose, Lacto-N-neo-

tetraose・ABO series・・・ A trisaccharide, B trisaccharide

O disaccharide

Each glycan is printed in triplicate at high and low concentrations.

Glycan structures (24 types)

・Ganglioside seriesGM3, GD3, GT3, GM2, GD2, GT2, GM1a, GM1b, GD1a, GD1b, GT1a, GT1c, GA1 (asialo GM1), Fucosyl GM1, GA2 (asialo GM2)

・Globo seriesGb3 (Pk antigen), Gb4 (P antigen), Gb5 (SSEA-3), Globo-H (SSEA-3), Globo-A, Globo-B, SSEA-4 tetraose, SSEA-4 hexaose, Forssman antigen

Each glycan is printed in triplicate at high and low concentrations.

GlycanArray I GlycanArray II

◆ Example of binding assay using GlycanArray I -Evaluation of binding specificity of lectins-

◆ Example of binding assay using GlycanArray II-Evaluation of binding specificity of Cholera Toxin Subunit B -

Figure 2.Cholera toxin subunit B is known to bind to

GM1a ganglioside on the surface of small-intestinal epithelial cell membrane. When fluorescently-labeled cholera toxin subunit B was reacted on GlycanArray 2, as expected, it bound specifically to GM1a and several structurally-similar glycolipid sugar chains. Fluorescent intensities were detected by Typhoon TRIO+ (GE Healthcare).

Figure 1.Binding specificities between immobilized glycans and lectins were

evaluated using GlycanArray I.After applying Biotin-AAL(10mg/mL) or Biotin-SNA(100mg/mL) onto

the slides, Cy3-Streptavidin was added. Fluorescent intensity on each spot was detected by ScanArrayLite(PerkinElmer).As expected, AAL lectin bound selectively to fucosylated glycans, and

SNA lectin bound to glycans with terminal a2-6Sia structure.

AAL lectin SNA lectin

[Reference]Oda M, Kabura M, Takagishi T, Suzue A, Tominaga K, Urano S, Nagahama M, Kobayashi K, Furukawa K, Furukawa K, Sakurai JClostridium perfringens alpha-toxin recognizes the GM1a/TrkA complexJ Biol Chem 287(39), 33070-33079 (2012)

Cat. No Product Qty/Cs

BS-X1731Z Glycan array I 1



BS-X1714Z Glycan Array II 1



SERVICESS-BIO specializes in the rapid and efficient analysis of both N-linked and O-linked glycans present on antibodies and other glycoproteins. Our proprietary GlycanMap® and GlycanMap Xpress® Services that combines BlotGlyco® bead-based glycan purification in a fully automated sample processing system with high-throughput MALDI-TOF mass spectrometry to generate rapid, reproducible glycan profiles. The resulting qualitative and quantitative information can be employed to achieve a variety of endpoints related to strain selection, fermentation and process development, formulation stability and product characterization. S-BIO’s services provide a new solution to accelerate your biotherapeutic development and biomarker discovery programs by enabling accelerated glycoform analysis across literally hundreds of samples.

GLYCAN ANALYSIS PRODUCTSWe recognize that many organizations prefer to perform analysis in-house for a variety of reasons. S-BIO offers its technology to customers in the form of BlotGlyco® beads and purification and labeling kits especially designed for glycan analysis. S-BIO also offers printed Glycan Array and Plates for binding assay.

GLYCAN ANALYSIS PRODUCTS AND SERVICES.S-BIO offers high-throughput analysis services and kits based our BlotGlyco® polymer beads for supplementary glycan purification.

glycan analysis - biogenuix.com · blotglyco®-glycan purification and labeling kit-1. blotglyco®...

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