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LecChip™ – Lectin Microarray | GK-LECCHIP

Cat. No.: GK-LECCHIP

1 pcs / € 705Please Inquire

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Glycan Profiling using Lectin Microarrays:
Therapeutic Areas
Assay Characteristics
  • Category number

    GK-LECCHIP

  • Method

    Lectin microarray detecting labeled carbohydrates/labeled cells

  • Sample type

    Purified and crude samples, e.g. cell/tissue extracts, living cells, virus particles, cell culture supernatants, serum, urine

  • Sample volume

    Cells: 3×106 cells

    Purified/crude sample: 20 µl (50 µg/ml protein in PBS)

  • Assay time

    Cells: ~ 1 h

    Purified/crude samples: overnight

  • Sensitivity

    LOD: 100 pg/ml glycoprotein, 100 pM glycan, 103 cells

  • Use

    Research use only.

  • Specificity

    N- and O-glycans; occurence is tested by binding to lectins

Product details

Product Overview

Lectin microarrays using LecChip™ allows the highly sensitive analysis of carbohydrates in various sample types (purified and crude samples, e.g. cell/tissue extracts, living cells, virus particles, cell culture supernatants, serum, urine). Compared to MS-based methods, it requires less sample processing steps and is about 100 times more sensitive. Although it cannot identify glycan structures perfectly, it is a powerful system to identify differences of glycan structures like glycan isomers in a small amount of sample. Most importantly, it is possible to simultaneously obtain data about O- as well as N-glycans.

Analysis of LecChip™ requires an evanescent-field fluorescence excitation scanner like GlycoStation™ or GlycoLite™2200.

Principle of the LecChip™

The principle of glycan structure profiling analysis using LecChip™ is based on the lectin to glycan binding affinity. Lectins are glycan binding proteins and each lectin will only bind to particular glycans. A LecChip™ contains 45 different lectins immobilized on a slide glass in an X-Y array. Cy3 labeled glycoproteins are applied to the LecChip™ surface which then bind to specific lectins on the chip. The LecChip™ is then analyzed with an evanescent-field fluorescence excitation scanner like GlycoStation™ or GlycoLite™2200 capable of generating an evanescent-field above the LecChip™ array. The Cy3 molecules attached to glycans that have successfully bound to lectins will fluoresce and the total LecChip™ fluorescence pattern can be captured with the scanner. Differential data analysis it best accomplished with the GlycoStation™ ToolsPro Ver.2.0 software.

The evanescent-field fluorescent excitation method used to excite the Cy3 marker on the glycans is an essential enabling technology as it allows for detection of very weak molecular interactions. Lectin-glycan interactions are known to be relatively weak compared to antigen-antibody and biotin-avidin interactions. If a washing process is applied to the LecChip™ to remove non-lectin binding redundant glycoproteins, many of the specifically bound glycans are also lost. Fortunately, evanescent-field fluorescence excitation allows for generation of lectin-glycan affinity data from unwashed samples by taking advantage of the higher signal strength of specifically bound glycans. The evanescent-field is formed on the surface of the LecChip™ when light enters into the slide glass from the sidewall and propagates through the glass. This is known as the principle of internal reflection mode. The evanescent field that is formed has a depth equal to the wavelength of the light and the field strength decreases exponentially with the distance away from the slide surface. Cy3-tagged glycans floating above the slide glass in a liquid phase exhibit a relatively low level of excitation as they are in the weakest part of the evanescent-field. By contrast, Cy3-tagged glycans that interact with the lectins located on the LecChip™ are contained in the stronger part of the evanescent field and are effectively excited. Hence, this technology allows monitoring of very weak molecular interactions directly from a liquid phase without washing of the sample.

Components

Contents

Description

Quantity

LecChip™

Glass slide with 45 different immobilized lectins*, spotted in triplicates

1 x chip

* Lectins spotted on LecChips™:



No.

Lectin (origin)

Reported glycan selectivity

1

LTL (Lotus tetragonolobus)

Fucα1-3(Galβ1-4)GlcNAc (Lewis x), Fucα1-2Galβ1-4GlcNAc (H-type 2)

2

PSA (Pisum sativum)

Fucα1-6GlcNAc (Core Fuc) , α-Man

3

LCA (Lens culinaris)

Fucα1-6GlcNAc (Core Fuc), α-Man

4

UEA-I (Ulex europaeus)

Fucα1-2Galβ1-4GlcNAc (H-type 2)

5

AOL (Aspergillus oryzae)

Fucα1-6GlcNAc (Core Fuc), Fucα1-2Galβ1-4GlcNAc (H-type 2)

6

AAL (Aleuria aurantia)

Fucα1-3(Galβ1-4)GlcNAc (Lewis x), Fucα1-6GlcNAc (Core Fuc)

7

MAL_I (Maackia amurensis)

Siaα2-3Galβ1-4GlcNAc

8

SNA (Sambucus nigra)

Siaα2-6Gal/GalNAc

9

SSA (Sambucus sieboldiana)

Siaα2-6Gal/GalNAc

10

TJA-I (Trichosanthes japonica)

Siaα2-6Gal/GalNAc, HSO3(-) -6Gal b1-4GlcNAc

11

PHAL (Phaseolus vulgaris)

tri/tetra-antennary complex-type N-glycan

12

ECA (Erythrina cristagalli)

Galβ1-4GlcNAc (up with increasing the number of terminal Gal), no affinity for fully sialylated N-type, fully agalactosylated N-type

13

RCA120 (Ricinus communis)

Galβ1-4GlcNAc (up with increasing the number of terminal Gal), Galb1-3Gal (weak), no affinity for agalactosylated N-type

14

PHAE (Phaseolus vulgaris)

bi-antennary complex-type N-glycan with outer Gal and bisecting GlcNAc, no affinity for fully sialylated N-type

15

DSA (Datura stramonium)

(GlcNAcβ1-4)n (Chitin), tri/tetra-antennary N-glycan

16

GSL-II (Griffonia simplicifolia)

agalactosylated tri/tetra antennary glycans, GlcNAc, no affinity for fully galactosylated or sialylated N-type

17

NPA (Narcissus pseudonarcissus)

High-Mannose including Manα1-6Man

18

ConA (Canavalia ensiformis)

High-Mannose including Manα1-6(Manα1-3)Man

19

GNA (Galanthus nivalis)

High-Mannose including Manα1-3Man

20

HHL (Hippeastrum hybrid)

High-Mannose including Manα1-3Man or Manα1-6Man

21

ACG (mushroom, Agrocybe cylindracea)

Gal b1-3Gal, Siaα2-3Galβ1-4GlcNAc

22

TxLCI (Tulipa gesneriana)

Manα1-3(Manα1-6)Man, bi/tri-antennary complex-type N-glycan, GalNAc

23

BPL (Bauhinia purpurea)

Galβ1-3GalNAc (up with Lewis x, down with Core Fuc), GalNAc

24

TJA-II (Tanthes japonica)

Fucα1-2Galβ1-> or GalNAcβ1-> groups at their non-reducing terminals

25

EEL (Euonymus europaeus)

Gala1-3Galb1-4GlcNAc, Fuca1-2Galb1-3GlcNAc (H antigen)

26

ABA (fungus, Agaricus bisporus)

Galβ1-3GalNAc, GlcNAc

27

LEL (tomato, Lycopersicon esculentum)

(GlcNAcβ1-4)n (Chitin), (Galb1-4GlcNAc)n (polylactosamine)

28

STL (potato, Solanum tuberosum)

(GlcNAcβ1-4)n (Chitin) oligosaccharide containing GlcNAc and MurNAc

29

UDA (Urtica dioica)

GlcNAcβ1-4GlcNAc (Chitin), High-Mannose (3 to High, up with increasing the number of Man)

30

PWM (pokeweed, Phytolacca Americana)

(GlcNAcβ1-4)n (Chitin)

31

Jacalin (Artocarpus integrifolia)

GlcNAcb1-3GalNAc (Core3), Siaa2-3Galb1-3GalNAc (sialyl T), Galb1-3GalNAc (T-antigen), a-GalNAc (Tn-antigen)

32

PNA (peanut, Arachis hypogaea)

Galβ1-3GalNAc

33

WFA (Wisteria floribunda)

GalNAcβ1-4GlcNAc (LacdiNAc), Galβ1-3(-6)GalNAc

34

ACA (Amaranthus caudatus)

Galβ1-3GalNAc (T-antigen), Siaa2-3Galb1- GalNAc (sialyl T)

35

MPA (Maclura pomifera)

a-GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen)

36

HPA (snail, Helix pomatia)

α-GalNAc

37

VVA (Vicia villosa)

GalNAcb1-4Gal, GalNAcb1-3Gal, a-GalNAc,

38

DBA (Dolichos biflorus)

Blood group A, GalNAcα1-3GalNAc, GalNAcb1-4(Siaa2-3)Galb1-4Glc (GM2)

39

SBA (soybean, Dolichos biflorus)

α- or β-linked GalNAc, Gala1-4Gal-Glc

40

Calsepa (Calystegia sepium)

Galactosylated bianntenary N-type with bisecting GlcNAc (galacto > agalacto, down with Core Fuc), High-Mannose (Man2–6)

41

PTL-I (Psophocarpus tetragonolobus)

α-GalNAc, Gala1-3(Fuca1-2)Gal (B-antigen)

42

MAH (Maackia amurensis)

Siaα2-3Galβ1-3(Siaα2-6)GalNAc (disialyl-T)

43

WGA (wheat germ, Triticum aestivum)

(GlcNAcβ1-4)n (Chitin), Hybrid type N-glycan, Sia

44

GSL-I A4 (Griffonia simplicifolia)

α-GalNAc

45

GSL-I B4 (Griffonia simplicifolia)

α-Gal
Instructions for use

Glycan Profiling of Crude Sample

1. Sample preparation and fractionation

 

1-1. Wash cells (5 × 106) by PBA several times and store at -80℃.
1-2. Fractionate the cells into membrane and cytoplasm fractions by using a commercial Kit

 

2. Cy3 labeling

 

2-1. Measure protein concentration with a commercial Micro BCA Protein Assay Reagent Kit (reaction time = 2 h).
2-2. Prepare 20 µl sample volume with a concentration of 50 µg/ml using PBS, then mix with Cy3 Mono-Reactive dye 100 µg labeling.
2-3. Incubate for 1 h in a dark place at R.T.
2-4. Add 300 µl TBS into a gel filtration column, then centrifuge at 1,500×g, for 1 min at 4℃.
2-5. Repeat two times.
2-6. Add the whole sample prepared in 2-3 and 25 µl TBS into the gel filtration column prepared at 2-5, then centrifuge at 1,500 × g, for 2 min, at 4℃, and remove excess Cy3.

 

3. Measurement

 

3-1. Dilute samples with a Probing Solution (in a range from 2µ g/mL to 31.25 ng/mL).
3-2. Wash LecChip™ with a Probing Solution three times, then apply samples into wells (100 µL/well).
3-3. Incubate LecChip™ at 20℃ over night.
3-4. Measure fluorescence patterns without any washing of LecChip™ by GlycoStation™ Reader 1200.
3-5. Analyse the results by Array-Pro™ Analyzer and GlycoStation™ Tools.

 

4. Protocol Booklet

 

Crude Sample

Glycan Profiling of Secreted Proteins

 

1. Enrichment of secreted proteins and buffer exchange

 

1-1. Collect 10 ml of culture supernatant and store it at -80℃.
1-2. Melt the sample from step 1-1, and apply it to a barrier filter. Centrifuge it at 4,000 × g, at 4℃, and thereby enrich the sample to less than 500 µl.
1-3. Add 14.5 ml of PBS to the sample. Centrifuge the sample at 4,000 × g, at 4℃, and enrich the sample to less than 500 µl (enrichment and buffer exchange).
1-4. Repeat step 1-3.
1-5. Add 14.5 ml of PBS to the 1-4 result. Centrifuge the sample at 4,000 × g, at 4℃, enrich the sample to less than 250 µL, and recover the sample.
1-6. Prepare 500 µl sample adding PBS to the 1-5 result.

 

2. Cy3 labeling

 

2-1. Measure protein concentration with a commercial Micro BCA Protein Assay Reagent Kit (reaction time = 2 h).
2-2. Prepare 20 µl sample volume with a concentration of 50 µg/ml using PBS, then mix with Cy3 Mono-Reactive dye 100 µg labeling.
2-3. Incubate for 1 h in a dark place at R.T.
2-4. Add 300 µl TBS into a gel filtration column, then centrifuge at 1,500×g, for 1 min at 4℃.
2-5. Repeat two times.
2-6. Add the whole sample prepared in 2-3 and 25 µl TBS into the gel filtration column prepared at 2-5, then centrifuge at 1,500 × g, for 2 min, at 4℃, and remove excess Cy3.

 

3. Measurement

 

3-1. Dilute samples with a Probing Solution (in a range from 2 µg/mL to 31.25 ng/mL).
3-2. Wash LecChip™ by a Probing Solution three times, then apply samples into wells (100 µl/well).
3-3. Incubate LecChip™ at 20℃ over night.
3-4. Measure fluorescence patterns without any washing of LecChip™ by GlycoStation™ Reader 1200.
3-5. Analyse the results by Array-Pro™ Analyzer and GlycoStation™ Tools.

 

4. Protocol Booklet

 

Secreted Proteins

 

Glycan Profiling of Living Cells

 

1. Collection of cell sample

 

1-1. Retrieve 3 × 106 cells and wash those with serum-free culture media.

 

2. Fluorescent labeling

 

2-1. Add 3 ml CellTracker™ to the cells that were suspended with serum-free culture media.
2-2. Stir the sample for 15 min at 37℃ in the dark.
2-3. Wash the cells with 1% BSA/PBS after the incubation.

 

3. Measurement

 

3-1. Dilute the dyed cells with 1% BSA/PBS. 
3-2. Wash the LecChip™ three times with Probing Solution, then add samples into wells（100 ml / well）.
3-3. Incubate the LecChip™ for 30 min at 4℃.
3-4. Put the incubated LecChip™ into the 50 ml tube that filled with PBS, then stand for 30 min with the well-side below. The cells that doesn't bind with the lectin are removed by gravity.
3-5. Scan the LecChip™ with GlycoStation™ Reader 1200.
3-6. Analyze the results with Array-Pro™ Analyzer and GlycoStation™ Tools.

 

4. Protocol Booklet

 

Living Cells

Background & Therapeutic Areas

Background

Glycosylation is a common posttranslational modification of proteins and influences parameters like protein folding, targeting, ligand recognition, stability, immunogenicity, biological activity, etc. Glycans play a significant biological role in mainly four medical areas: cancer, immunology, infectious diseases and regenerative medicine. Here, a quick, simple and highly sensitive method for glycan profile analysis can accelerate the development of advanced biomarkers, therapeutic proteins, probiotic methodologies as well as the characterization of stem cell (iPS, ES, and MSC).

Applications

  • Detection of Changes in Glycosylation Pattern

  • Supporting Quality Control Activities (Batch-to-Batch Variation)

  • Comparability Testing of Biosimilars

  • Monitoring of Biologicals (e.g. Protein Drugs)

  • Host Cell Characterization

  • Glyco-Biomarker Discovery

  • Characterization of Stem Cells & Differentiated Cells

  • Advanced Histochemistry (e.g. Marker Definition)

  • Production Process Optimization (IPC testing)

Glycan Profiling & Lectin Microarray – Applications

Lectin Microarray and Therapeutic Proteins

FDA publication: Glycan analysis of therapeutic glycoproteins. L. Zhang, S. Luo, and B. Zhang, mAbs, 2015; 8:2, 205-215.

FDA publication: The use of lectin microarray for assessing glycosylation of therapeutic proteins. L. Zhang, S. Luo, and B. Zhang, mAbs, 2015; 3: 524-535.

Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC. Roucka, Markus, Klaus Zimmermann, Markus Fido, und Andreas Nechansky. Microarrays, 2017; 6, 1  

Glycosylation Pattern of Biotechnologically Produced Proteins - Lectin Array Technology as a Versatile Tool for Screening? Zimmermann, Fido, und Roucka. Medical Research Archives, 2018; 6: 3.

Exosomes

Glycan profiling analysis using evanescent-field fluorescence-assisted lectin array: Importance of sugar recognition for cellular uptake of exosomes from mesenchymal stem cells. Shimoda A, Tahara Y, Sawada SI, Sasaki Y, Akiyoshi K. Biochem Biophys Res Commun. 2017; 23;491(3):701-707.

Diabetic Nephropathy

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray. Kentaro Inoue,Jun Wada ,Jun Eguchi,Atsuko Nakatsuka,Sanae Teshigawara,Kazutoshi Murakami,Daisuke Ogawa,Takahiro Terami,Akihiro Katayama,Atsuhito Tone,Izumi Iseda,Kazuyuki Hida,Masao Yamada,Tomohisa Ogawa,Hirofumi Makino. Plos One, 2013; 8: 10:  e77118.

Cancer

A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin. Yukinari Kato & Mika Kato Kaneko. Scientific Reports; 2014; 4: 5924.  

Lectin microarray technology identifies specific lectins related to lymph node metastasis of advanced gastric cancer. Yamashita K, Kuno A, Matsuda A, Ikehata Y, Katada N, Hirabayashi J, Narimatsu H, Watanabe M. Gastric Cancer, 2016; 9(2):531-542.

Lectin Microarray-Based Sero-Biomarker Verification Targeting Aberrant O-Linked Glycosylation on Mucin 1. Matsuda A, Kuno A, Nakagawa T, Ikehara Y, Irimura T, Yamamoto M, Nakanuma Y, Miyoshi E, Nakamori S, Nakanishi H, Viwatthanasittiphong C, Srivatanakul P, Miwa M, Shoda J, Narimatsu H. Anal Chem, 2015; 21;87(14):7274-81.

Retinal degradation:

Lectin microarray profiling and relative quantification of glycome associated with proteins of neonatal wt and rd1 mice retinae. Ahuja S. Invest Ophthalmol Vis Sci, 2013: 7;54(5):3272-80.

Tissue

A standardized method for lectin microarray-based tissue glycome mapping. Zou X, Yoshida M, Nagai-Okatani C, Iwaki J, Matsuda A, Tan B, Hagiwara K, Sato T, Itakura Y, Noro E, Kaji H, Toyoda M, Zhang Y, Narimatsu H, Kuno A. Sci Rep, 2017; 6;7:43560.

Immune response

Fucosylation is associated with the malignant transformation of intraductal papillary mucinous neoplasms: a lectin microarray-based study. Watanabe K, Ohta M, Yada K, Komori Y, Iwashita Y, Kashima K, Inomata M. Surg Today, 2016; 46(10):1217-23.

Liver disease

A serum “sweet-doughnut” protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis. Atsushi Kuno, Yuzuru Ikehara, Yasuhito Tanaka, Kiyoaki Ito, Atsushi Matsuda, Satoru Sekiya, Shuhei Hige, Michiie Sakamoto, Masayoshi Kage, Masashi Mizokami & Hisashi Narimatsu. Scientific Reports, 2013; 3: 1065.  

Dysplasia in Barrett's esophagus

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus. Bird-Lieberman EL, Neves AA, Lao-Sirieix P, O'Donovan M, Novelli M, Lovat LB, Eng WS, Mahal LK, Brindle KM, Fitzgerald RC. Nat Med, 2012; 15;18(2):315-21.

Review-Applications

Lectin microarrays: concept, principle and applications.Jun Hirabayashi, Masao Yamada, Atsushi Kuno and  Hiroaki Tateno. Chem Soc Rev, 2013; 42: 4443-4458.

Lectin-based structural glycomics: a practical approach to complex glycans. Hirabayashi J, Kuno A, Tateno H. Electrophoresis, 2011; 32(10):1118-28.

Citations


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