RayBiotech Lectin Array 95--Detect glycan profiles using 95 lectins

User Manual
July 11th, 2022

Cat # GA-Lectin-95

RayBiotech, Inc.
We Provide You With Excellent
Protein Array Systems and Service
Tel:(Toll Free) 1-888-494-8555 or 770-729-2992; Fax: 770-206-2393;
Website:www.RayBiotech.com Email: info@RayBiotech.com

Lectins printed onslides (95)AAA, AAL, ABA, ABL, ACG, ACL, AMA, ASA, BanLec, BC2L-A, BC2LCN,
BPA, CA, CAA, Calsepa, CGL2, CNL, Con A, CPA, CSA, DBA, Discoidin I,
Discoidin II, DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S,
Gal7-S, Gal9, GHA, GNA, GRFT, GS-I, GS-II, HAA, HHA, HMA, IRA,
Jacalin, LAL, LBA, LCA, LEA, Lentil, Lotus, LPA, LSL-N, MAA, Malectin,
MNA-G, MNA-M, MOA, MPL, NPA, Orysata, PA-IIL, PA-IL, PALa, PHA-E,
PHA-L, PHA-P, PNA, PPL, PSA, PSL1a, PTL-1, PTL-2, PWA, RCA-120, RCA60, RPA, RS-Fuc, SAMB, SBA, SHA, SJA, SNA-I, SNA-II, SSA, STL, TL, UDA,
UEA-I, UEA-II, VFA, VRA, VVA, VVA-M, WFA, WGA

FormatOne standard glass slide is spotted with 12 wells ofidentical lectin sub-arrays. Each lectin is printed in
duplicate on every sub-array

Detection Method Fluorescence with laser scanner: Cy3 equivalent dye
Sample Volume

50 – 100 l per array

Reproducibility

CV <20%

Assay duration

6 hrs

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RayBiotech Lectin Array 95 Kit

TABLE OF CONTENTS
I.

Overview………………………………………………………………………………………………………………………………..………………….… 1
A. Introduction…........................................................................................................................................3
B. How It Works…………………………………………………………………………………………………………………………….…...4

II.

Materials Provided……………………………………………………………………………………………….………………..…….. 5

III.

Glass Slide Layout…………………………………………………………………………………………………..………………………..6

IV.

General Considerations………………………………………………………………………………………………….….…… 6
A. Label-Based vs. Sandwich-Based Method…………………………………………… 6
B. Preparation of Samples……………………………………………………………………………………………….… 7
C. Handling Glass Slides…………………………………………………………………………………………..……….…….7
D. Incubation…………………………………………………………………………………………………………………………………….…… 7

V.

Protocol……………………………………………………………………………………………………………………………………………………….… 8
A. Dialysis of Sample…………………………………………………….……………………………………………………………. 8
B. Biotin-labeling Sample………………………………………………………….………………………………………….9
C. Dry the Glass Slide………………………………………………………………………………………………………….………10
D. Blocking and Incubation…………………………………………………………………………………….…………. 10
E. Incubation with Cy3 Equivalent Dye-Streptavidin ……………...… 11
F. Fluorescence Detection …………..……......……..……..……..……..……..……..……..……..……..…….. 12
G. Data Analysis ………………………………………………………………………………………….…………………………………… 13
H. Normalization of Array Data ………………………………………………………………..……………….. 13

VI.

Lectin Array 95 Map...……………………………………………………………….………………………………………………… 14

VII. Lectin Array 95 Key……………………………………………………………………………………………………………….

14

VIII. Application 1- Detection of Glycans on HRP…………………………………..………...16
IX.

Application 2- Profile of Serum Sample……………………………………………….……..….. 18

X.

Other Applications………………………………………………………………………………………………………………….…….. 18

XI.

Troubleshooting Guide…………………………………………………………………………………………………………....192

RayBiotech Lectin Array 95 Kit

I. Overview
A. Introduction
Glycocalyx, literally meaning ‘sugar coat’, is an extracellular polymeric coating
surrounding many prokaryotic and eukaryotic cells consisting ofglycoproteins, glycolipids, proteoglycans and glycosaminoglycans. The
constituents of the glycocalyx play an important role in the process of cellsignaling, virus transfection, and immunity. However, detection tools for the
research of glycobiology are currently in very limited supply.
RayBiotech, pioneered the development of antibody arrays, which are now
widely applied in the research community with hundreds of peer reviewed
publications such as in Cell and Nature. Taking advantage of advancements
in microarray technology developed for antibody arrays, we recently
developed lectin arrays to help researchers: 1) identify and profile the glycans
in their samples; 2) determine whether their biomarker of interest has glycan
moieties, and; 3) find specific glycan binding ligands in biological samples.
Lectins are glycan-binding proteins which have been purified from trees,beans and some fruits. They are highly specific for a given glycan based on
their sequence and the different sugar unit structures the glycan contains.
Based on the previously launched Lectin Array 40 and Lectin Array 70,
RayBiotech now introduces the Lectin Array 95, which is currently the largest
commercially available lectin array. The Lectin Array 95 consists of a standard
glass slide spotted with 12 identical lectin arrays (1 per well), each containing
95 unique lectins spotted in duplicate, plus positive controls. The slide comeswith a 16-well removable gasket which allows for the processing of 12
samples using one slide. Four slides can be nested into a tray, which matchesa standard microplate and allows for the automated robotic high-throughput
processing of 32 arrays simultaneously. The RayBio® Lectin Array 95 arrayprovides a powerful new tool for glycosylation determination, drug
discovery, and biomarker development, all while requiring limited sample
volumes.

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RayBiotech Lectin Array 95 Kit

B. How it Works

4
RayBiotech Lectin Array 95 Kit

II. Materials Provided
Upon receipt, all components of the RayBiotech Lectin Array 95 kit should be
stored at -20°C. After initial use, remaining reagents should be stored at 4°C
to avoid repeated freeze-thaw cycles and may be stored for up to 3 months(Labeling Reagent, Item B, should be prepared fresh each time before use).
Unused glass slides should be kept at -20°C and repeated freeze-thaw cycles
should be avoided (slides may be stored for up to 6 months). The entire kit
should be used within 6 months of purchase.

Components
Item Description
A
Dialysis Vials and Floating Dialysis Rack
B
D
E
F
G
H
I
J
K
M
N

Labeling Reagent
Stop Solution
Lectin Array Glass Slide Assembly
Sample Diluent
20X Wash Buffer I
20X Wash Buffer II
Cy3 equivalent dye-conjugatedStreptavidin
Adhesive device sealer
Labeling Buffer
Slide Washer/Dryer
Manual

1-Slide kit
24 vials/2
racks
4
1
1
1
1
1
1

2-Slide kit
48 vials/4
racks
8
2
2
1
1
1
2

4-Slide kit
96 vials/8
racks
16
4
4
2
2
2
4

2
1
1
1

4
2
1
1

8
4
2
1

Additional Materials Required• Detection antibodies of interest (For sandwich-based method only)• Orbital shaker• Laser scanner for fluorescence detection• Aluminum foil
• 1.5ml Polypropylene microcentrifuge tubes• KCl, NaCl, KH2PO4 and Na2HPO4 (For label-based method only)• Plastic or glass containers, beaker, stir plate and stir bar• Pipettors, pipette tips, ddH2O and other common lab consumables
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RayBiotech Lectin Array 95 Kit

III. Glass Slide Layout
The Lectin Glass Slide includes 12 wells that are printed with the lectin array,and 4 wells that have been left blank. Please use the diagram below to ensureloading of samples in the correct wells.

Printed Lectin Arrays

Blank Wells

IV. General Considerations
A. Label-Based vs. Sandwich-Based Method
The RayBiotech Lectin Array 95 Kit can be used with either a label-based
method or as a sandwich-based method.
• The label-based method is used to biotinylate samples containing
proteoglycans and glycoproteins for direct detection on the array via a
Cy3 equivalent dye-conjugated Biotin-Streptavidin complex. A
complete protocol and the primary materials for this procedure areincluded with the kit.
• The sandwich-based method is used for antibody detection of
glycocalyx elements (glycolipids, glycoproteins, etc.) captured on thearray. The user will need to supply the labeled reporter antibodies
specific for the glycocalyx elements of interest. An example protocol
for this procedure with a general “Antibody Cocktail” is included in this6
RayBiotech Lectin Array 95 Kitmanual. Specific antibody concentrations and conditions will need to
be determined by the end user.
B. Preparation of Samples• Use serum-free conditioned media if possible.
• If serum-containing conditioned media is required, it is highly
recommended that complete medium be used as a control since many
types of sera contain glycocalyx elements.
• We recommend the following parameters for your samples:o 50 to 200 μl of original or diluted serum, plasma, cell culture
media, or other body fluid, or 50-100 μl of cell or tissue lysateswith 1-2 mg/ml total protein concentration.
Note: If you experience high background or the readings exceed the detection
range, further dilution of your sample is recommended.
C. Handling Glass Slides• Do not touch the surface of the slides, as the microarray slides are very
sensitive. Hold the slides by the edges only.
• Handle all buffers and slides with latex-free gloves.
• Handle the glass slide in a clean environment.
• Permanent marker ink can significantly interfere with fluorescent signal
detection. Never mark anywhere on the front (arrayed) side of theslide. It’s best to avoid using marker completely, however if you needto number the slide, please add a small mark only on the back of theslide along the top or bottom edge using a green or blue ultra-fine point
Sharpie® brand marker, only after the slide is completely dry.
D. Incubation
A. Completely cover array area with sample or buffer during incubation.
B. Avoid foaming during incubation steps.
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RayBiotech Lectin Array 95 Kit

C. Perform all incubation and wash steps under gentle rotation.
D. Cover the incubation chamber with the adhesive film during incubation
to prevent evaporation, particularly when incubation is more than 2hours or <70 l of sample or reagent is used.
E. Several incubation steps such as step 6 (blocking), step 7 (sample
incubation), step 10 (detection antibody incubation), or step 13 (Cy3equivalent dye-streptavidin incubation) may be done overnight at 40C.
Please make sure to cover the incubation chamber tightly to prevent
evaporation.

V. Protocol
READ ENTIRE PROTOCOL BEFORE STARTING
A. Dialysis of Sample
Note: For the Sandwich-based protocol start at C. Drying the Glass Slide,step 8, on page 10. Do not do steps 1-7.
Note: Samples must be dialyzed prior to biotin-labeling (Steps 5–7).
1. Prepare enough dialysis buffer (1X PBS, pH=8.0) for all dialysis steps
herein and after. To prepare 1 L dialysis buffer, dissolve 0.2 g KCl, 8 g
NaCl, 0.2 g KH2PO4 and 1.15 g Na2HPO4 in 800 ml ddH2O. Adjust pH=8.0
with 1M NaOH and adjust final volume to 1000 ml with ddH2O.
2. Add each sample into a separate Dialysis Vial (Item A). Load 200 μl cell
culture supernatant or 100 μl cell lysate or tissue lysate (1-2 mg/ml total
protein) or 20 μl serum or plasma + 80 μl 1X PBS, pH=8 (5-fold dilution).
Carefully place Dialysis Vials into Floating Dialysis Rack.
Note: If the samples appear to be cloudy, transfer the samples to a clean tube,
centrifuge at 13,000 rpm for 20 minutes at 2-8°C. If the samples are still
not clear, store them at -20°C for 20 minutes. Remove from the freezer,
immediately centrifuge at 13,000 rpm for 20 minutes at 2-8°C.

8RayBiotech Lectin Array 95 Kit

3. Place Floating Dialysis Rack into at least 500 ml dialysis buffer in alarge beaker. For more than 2 samples, make certain to use at least
300 ml dialysis buffer for each sample (more buffer will improve theefficiency of dialysis). Place beaker on a stir plate and dialyze for atleast 3 hours at 4°C, stirring buffer gently. Then exchange the dialysis
buffer and repeat dialysis for another 3 hours at 4°C. Transfer dialyzed
sample to a clean eppendorf tube. Spin dialyzed samples for 5 min at
10,000 rpm to remove any particulates or precipitants, and thentransfer the supernatants to a clean tube.
Note: The sample volume may change during dialysis.
Note: Dialysis procedure may proceed overnight.
Note: Determine the total protein concentration for cell culture supernatantsor cell/tissue lysate after dialysis procedure (Step 3). We recommended
using a BCA total protein assay (eg, Pierce, Catalog # 23227).
B. Biotin Labeling of Sample
Note: Amines (e.g., Tris, glycine) and azides quench the biotinylation reaction.
Avoid contaminating samples with these chemicals prior tobiotinylation.
4. Immediately before use, prepare 1X Labeling Reagent. Briefly spin down
the Labeling Reagent tube (Item B). Add 100 µl 1X PBS into the tube,
pipette up and down or vortex slightly to dissolve the lyophilized
reagent.
5. Add 1X Labeling Reagent to dialyzed samples.
a. For labeling cell culture supernatants: transfer 180 μl dialyzed
sample into a new tube. Add 36 μl of 1X Labeling Reagent Solution
per 1 mg total protein in dialyzed cell culture supernatant. Mix
well. For example, if sample’s total protein concentration is 0.5
mg/ml you need to add 3.24 µl 1X Labeling Reagent to the tube of
180 μl dialyzed sample.

9
RayBiotech Lectin Array 95 Kit

b. For labeling serum or plasma: Add 22 μl of 1X Labeling Reagent
Solution into a new tube containing 35 μl dialyzed serum or
plasma sample and 155 μl Labeling Buffer (Item K).
c. For labeling cell or tissue lysates: transfer 30 µg (for example, 15
μl of 2 mg/ml) cell or tissue lysates into a tube and add Labeling
Buffer (Item K) for a total volume of 300 μl. Then add 3.3 μl of 1X
Labeling Reagent Solution.
Note: To normalize serum/plasma or cell/tissue lysate concentrations duringbiotinylation, measure sample volume before and after dialysis. Thenadjust the volumes of dialyzed serum/plasma or cell/tissue lysates andLabeling Buffer to compensate.
6. Incubate the reaction solution at room temperature with gentle rocking
or shaking for 30 min. Mix the reaction solution by gently tapping thetube every 5 min.
7. Add 3 μl Stop Solution (Item D) into each reaction tube and immediately
dialyze as directed in Step 3.
Note: Biotinylated samples can be stored at -20°C or -80°C until you are readyto proceed with the assay.
C. Dry the Glass Slide
8. Take out the bag containing the glass slide from the box, and let theslide equilibrate to room temperature inside the sealed plastic bag for
20-30 minutes. Then, remove slide from the plastic bag; peel off thecover film, and let it air dry at room temperature for another 1-2 hours.
Note: Incomplete drying of slides before use may cause the formation of“comet tails”.
D. Blocking and Incubation
9. Add 100μl Sample Diluent (Item F) into each well and incubate at room
temperature for 30 min to block slides.
10
RayBiotech Lectin Array 95 Kit10. Immediately prior to sample incubation, spin biotin-labeled samples for
5 minutes at 10,000 rpm to remove any particulates or precipitates.
Dilute samples with Sample Diluent. Recommended dilution of thebiotin-labeled samples is 2-10 fold for cell culture supernatants, 20-100
fold for serum/plasma and 30-100 fold cell/tissue lysate, however,
optimization is recommended to do for the best results.
11. Decant buffer from each well. Add 100μl of sample to each well. Incubate
arrays at room temperature for 1-2 hours. (Longer incubation time ispreferable if higher signal intensity is desired)
Note: We recommend using 50 to 100 μl of original or diluted serum, plasma,conditioned media, or other body fluid, or 50-500 μg/ml of protein for celland tissue lysates. Cover the incubation chamber with adhesive film
during incubation if less than 70 ul of sample or reagent is used.
Note: This step may be done overnight at 40C for highest intensities.
Note: For the Sandwich-based protocol, it’s recommended to do optimization
to determine appropriate dilution of non-biotinylated samples for
incubation.
12. Wash:a. Calculate the amounts of 1x Wash Buffers I & II that are neededfor each step of the protocol. Separately dilute required amountsof 20x Wash Buffer I and 20x Wash Buffer II with ddH2O to 1x
concentration. For example if 12 ml of 1x Wash Buffer I is needed
then 600 μl of 20x Wash Buffer I would be diluted to a final volumeof 12 ml.
b. Decant the samples from each well, and wash each well 5 times (5min each) with 150 μl of 1x Wash Buffer I at room temperature
with gentle shaking. Completely remove wash buffer between
each wash step.
c. (Optional for Cell and Tissue Lysates) Put the glass slide with frame
into a box with 1x Wash Buffer I (cover the whole glass slide and11
RayBiotech Lectin Array 95 Kit

frame with Wash Buffer I), and wash at room temperature withgentle shaking for 20 min.
d. Decant the 1x Wash Buffer I from each well, wash 2 times (5 mineach) with 150 μl of 1x Wash Buffer II at room temperature withgentle shaking. Completely remove wash buffer between each
wash step.
Note: Incomplete removal of the wash buffer after each wash step may cause“dark spots”. (Background signal is higher than that of the spot.)
E. Incubation with Cy3 Equivalent Dye-Streptavidin
Note: For the Sandwich-based protocol, follow below steps after
secondary antibody incubation if biotinylated secondary antibody is used.
If fluorescence conjugated secondary antibody is used, skip steps 13-15
and continue from step 16 after incubation of secondary antibody.
Appropriate dilution of secondary antibody should be determined before
incubation.
13. Briefly spin down the Cy3 equivalent dye-conjugated streptavidin
tube.
14. Add 1.4 ml of Sample Diluent to Cy3 equivalent dye-conjugatedstreptavidin tube. Mix gently.
15. Add 80 μl of Cy3 equivalent dye-conjugated streptavidin to each well.
Cover the slide with aluminum foil to avoid exposure to light or incubate
in dark room. Incubate at room temperature for 1 hour.
16. Decant the samples from each well, and wash 5 times with 150 μl of
1x Wash Buffer I at room temperature with gentle shaking. Completely
remove wash buffer after each wash step.
F. Fluorescence Detection
17. Disassemble the slide assembly by pushing clips outward from theslide side. Carefully remove the slide from the gasket.
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RayBiotech Lectin Array 95 Kit

Note: Be careful not to touch thesurface of the array.
18. Place the slide in the slide
Washer/Dryer (a 4-slide holder/centrifuge tube), add enough 1x Wash
Buffer I (about 30 ml) to cover the whole slide, and then gently shake atroom temperature for 15 minutes. Decant Wash Buffer I. Wash with 1x
Wash Buffer II (about 30 ml) with gentle shaking at room temperaturefor 5 minutes.
19. Remove liquid droplets completely by one of the following ways:i.

Put the glass slide into the Slide Washer/Dryer, and dry the glass
slide by centrifuging at 1,000 rpm for 3 minutes without cap.

ii.

Or, dry the glass slide by a compressed N2 stream.

iii.

Or gently apply suction with a pipette to remove water droplets.
Do not touch the sub-array areas, only the sides of the slide.

20. Imaging: The signals can be visualized through use of a laser scanner
equipped with a Cy3 wavelength such as Axon GenePix. Make sure thatthe signal from the spot containing the highest concentration receives
the highest possible reading, yet remains unsaturated.
Note: If the signal intensity for different lectins vary greatly in the samearray, we recommend using multiple scans, with a higher PMT for low
signal lectins, and a low PMT for high signal lectins.
G. Data Analysis
21. Data extraction can be done using the GAL file that is specific for thisarray, along with the microarray software commonly available in mostmicroarray laser scanners (GenePix, ScanArray Express, etc.). GAL files
can be found on our website here.
www.RayBiotech.com/Gal-Files.html.
H. Normalization of Array Data
13
RayBiotech Lectin Array 95 Kit22. To normalize signal intensity data, one sub-array is defined as"reference" to which the other arrays are normalized. This choice can bearbitrary. For example, in our Analysis Tool Software, the array
represented by data entered in the left-most column each worksheet isthe default "reference array."
You can calculate the normalized values as follows:
X(Ny) = X(y) * P1/P(y)Where:
P1 = mean signal intensity of POS spots on reference array
P(y) = mean signal intensity of POS spots on Array "y"X(y) = mean signal intensity for spot "X" on Array "y"X(Ny)= normalized signal intensity for spot "X" on Array "y"VI. Lectin Array 95 Map
1
1 POS1POS2
2 POS1POS2
3 BC2L-A
BC2LCN
4 BC2L-A
BC2LCN
5 DISCOIDIN IIDSA
6 DISCOIDIN IIDSA
7 GNA
GRFT
8 GNA
GRFT
9 LENTIL
Lotus
10 LENTIL
Lotus
11 PA-IL
PALa
12 PA-IL
PALa
13 RCA 60
RPA
14 RCA 60
RPA
15 UEA I
UEA-II
16 UEA I
UEA-II

2

3
POS3
POS3
BPA
BPA
ECA
ECA
GS-I
GS-I
LPA
LPA
PHA-E
PHA-E
RS-FUC
RS-FUC
VFA
VFA

4
NEG
NEG
CA
CA
EEL
EEL
GS-II
GS-II
LSL-N
LSL-N
PHA-L
PHA-L
SAMB
SAMB
VRA
VRA

5
AAA
AAA
CAA
CAA
F17AG
F17AG
HAA
HAA
MAA
MAA
PHA-P
PHA-P
SBA
SBA
VVA
VVA

6
AAL
AAL
CALSEPACALSEPA
GAL1
GAL1
HHA
HHA
MALECTIN
MALECTIN
PNA
PNA
SHA
SHA
VVA-M
VVA-M

7
ABA
ABA
CGL2
CGL2
GAL1-SGAL1-S
HMA
HMA
MNA-G
MNA-G
PPL
PPL
SJA
SJA
WFA
WFA

8
ABL
ABL
CNL
CNL
GAL2
GAL2
IRA
IRA
MNA-M
MNA-M
PSA
PSA
SNA-I
SNA-I
WGA
WGA

9
ACG
ACG
Con A
Con A
GAL3
GAL3
Jacalin
Jacalin
MOA
MOA
PSL1A
PSL1A
SNA-II
SNA-II
NEG
NEG

10
ACL
ACL
CPA
CPA
GAL3C-SGAL3C-S
LAL
LAL
MPL
MPL
PTL-1
PTL-1
SSA
SSA
NEG
NEG

11
AMA
AMA
CSA
CSA
GAL7-S
GAL7-S
LBA
LBA
NPA
NPA
PTL-2
PTL-2
STL
STL
POS3
POS3

12
ASA
ASA
DBA
DBA
GAL9
GAL9
LcH A
LcH A
ORYSATAORYSATA
PWA
PWA
TL
TL
POS2
POS2

13
BANLECBANLECDISCOIDIN IDISCOIDIN I
GHA
GHA
LEA
LEA
PA-IIL
PA-IILRCA 120
RCA 120
UDA
UDA
POS1
POS1

VII. Lectin Array 95 Key

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RayBiotech Lectin Array 95 Kit

Lectins
1 Anguilla anguilla
2 Aleuria aurantia
3 Agaricus bisporus lectin

Abbreviation Source
AAA
Anguilla anguilla (Fresh Water Eel)
AAL
Aleuria aurantia mushrooms
ABA
Agaricus bisporus

4 Agaricus bisporus lectin

ABL

5 Agrocybe cylindracea lectin
6 Amaranthus caudatus
7 Arum maculatum lectin
8 Allium sativum

ACG

9 Musa acuminata lectin
10 Burkholderia cenocepacia lectin

BanLec11 Burkholderia cenocepacia lectin
12 Bauhinia purpurea
13 Colchicum autumnale
14 Caragana arborescens
15 Calystegia sepium lectin
16 Coprinopsis cinerea lectin
17 Clitocybe nebularis lectin
18 Coanavalin A
19 Cicer arietinum lectin
20 Cytisus scoparius lectin
21 Dolichos biflorus

ACL, ACA
AMA
ASA

BC2L-A

Carbonhydrate specificity
αFuc
Fucα6GlcNAc
Galactose (β1,3) N-Acetylgalactosamine
galactose-β-1,3-N-acetylgalactosamine, galactose-βAgaricus bisporus (White button mushroom) 1,3-N-acetylglucosamine
E. coli expressed Agrocybe cylindracea
galectin lectin
α2-3 Sialic Acid
Amaranthus caudatus seeds
Galβ3GalNAc
Arum maculatum (Lords and Ladies)
MannoseAllium sativum agglutinin (Garlic)
αMan
Mannose, Glucose, branched high-mannose
E. coli expressed Musa acuminata
E. coli expressed Burkholderia cenocepacia

BC2LCN（AiLecS1)
E. coli expressed Burkholderia cenocepacia
BPA, BLP
Bauhinia purpurea alba (Camel's Foot Tree)CA
Colchicum autumnale
CAA
Caragana arborescens (pea tree)
Calsepa
E. coli expressed Calystegia sepium
CGL2
CNL
Con A
CPA
CSA
DBA

E. coli expressed Coprinopsis cinerea
E. coli expressed Clitocybe nebularis
Coanavalia ensformis (Jack Beans) seeds
Cicer arietinum (chick pea)
Scotch broom
Dolichos biflorus (Horse Gram) seeds

containing α1,3-glycoside bondHigh-mannose
Fucα1-2Galβ1-3GalNAc (H type 3), Fucα1-2Galβ1-3GlcNAc(H type 1)Galβ3GalNAc
Lactose > N-Acetylgalactosamine > Galactose

N-Acetylgalactosamine
High-mannose
βGal, GalNAcα1-3Gal (Blood Group A), Galα1-3Gal(BloodGroup
B)
α/βGalNAc,
GalNAcβ1-4GlcNAc,
GalNAcα1-3[Fucα12]Galβ1-4GlcNAc (Blood Group A)

αMan, αGlc
FetuinN-Acetylgalactosamine
αGalNAc

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RayBiotech Lectin Array 95 Kit

Lectins
22 Dictyostelium discoideum lectin

Abbreviation Source
Discoidin I
E. coli expressed Dictyostelium discoideum

23 Dictyostelium discoideum lectin

Discoidin II

24 Datura stramonium

DSA, DSL

25 Erythrina cristagalli
26 Eunonymus europaeus
27 E. coli lectin
28 Pure Helix aspersa lectin
29 Homarus americanus lectin
Human galectin1 lectin (stable
30 form)
31 Human galectin1-S lectin
32 Human galectin2 lectin
Human galectin3 lectin (full33 length)
34 Human galectin 3C-S lectin
35 Human galectin7-S lectin
Human galectin9 lectin (Stable
36 Form)
37 Glechoma Hederacea lectin
38 Galanthus nivalis
39 Griffithia sp. Lectin
Griffonia (Banderaea)
40 simplicifolia I
41 Griffonia (Brandeiraea)
42 Hippeastrum hybrid
43 Iris hybrid lectin
44 Jacalin
45 Laburnum anagyroides lectin
46 Phaseolus lunatus
47 Lens Culinaris
48 Lycopersicon esculentum

ECA, ECL

49 Lentil lectin
50 Limulus polyphemus

Lentil51 Lotus tetragonolobus
52 Laetiporus sulphureus lectin
53 Maackia amurensis I
54 Human malectin lectin
55 Pure Morniga G lectin
56 Morniga M Lectin
57 Marasmius oreades lectin
58 Maclura pomifera
59 Narcissus pseudonarcissus
60 Oryza sative lectin
61 Pseudomonas aeruginosa lectin
62 Pseudomonas aeruginosa lectin
63 Phleb odium aureum lectin
Phaseolus vulgaris
64 Erythroagglutinin
65 Leucoagglutinin

EEL
F17AG
HAA
HMA
Gal1
Gal1-S

Gal2
Gal3
Gal3C-S

Gal7-S
Gal9
GHA
GNA, GNL
GRFT
GS-I, GSL-II,
BSL-I
GS-II, GSL-II,
BSL-II
HHA, HHL, AL

E. coli expressed Dictyostelium discoideum
Datura stramonium (Thorn Apple, Jimson
Weed) seeds
Erythrina cristagalli (Coral Tree) seeds
Eunonymus europaeus (Spindle Tree) seeds
E. coli expressed E. coli
Garden Snail
Homarus americanus (California lobster)E. coli expressed human galectin1 (stableform)
E. coli expressed human galectin1-S
E. coli expressed human galectin2

LcH, LCA
LEA, LEL, TL

Gal, LacNAc, Asialoglycans, Gal/GalNAcβ14GlcNAcβ1-6Gal/GalNAc(GlcNAc) 2-4
Galβ4GlcNAc
Galα3Gal
GlcNAcN-Acetylgalactosamine
N-Acetylneuraminic acid, N-Acetylgalactosamine
branched LacNAc, Gal
branched LacNAc
GalNAcα1-3Gal (Blood Group A), branched LacNAc

E. coli expressed human galectin9
Glechoma hederacea (ground ivy)
Galanthus nivalis (Snowdrop) bulbs
E. coli expressed Griffithia sp.

poly LacNAc, GalNAcα1-3Gal (Blood Group A)
Gal, methyl a-D-galactopyranoside, GalNAc
αMan
High-mannose

Griffonia (Banderaea) simplicifolia seeds
Griffonia (Banderaea) simplicifolia seeds
Hippeastrum hybrid (Amaryllis) bulbs

αGal, α3GalNAc
α or βGlcNAc
αMan
N-Acetyl-D-Galactosamine
Galβ3GalNAc
a-Me-L-Fucose among monosacchrides
GalNAcα(1,3)[αFuc(1,2]Gal
αMan, αGlc
(GlcNAc) 2-4
D-Mannose, D-glucose
Sialic Acid (N-Acetylneuraminic acid)

Artocarpus integrifolia (Jackfruit) seeds

LAL
LBA

αGalNAc (Tn antigen), LacNAc

E. coli expressed Human galectin3(full-length) poly LacNAc
E. coli expressed Human galectin 3C-S
poly LacNAc
E. coli expressed Human galectin7-S
Galβ1-3GlcNAc

IRA
Jacalin, AIL

Carbonhydrate specificity

Phaseolus lunatus (Lima Bean) seeds
Lens culinaris (lenti I) seeds
Lycopersicon esculentum (tomato) fruit

Lens culinaris seeds
lris hybrid (Dutch Iris)
Lotus tetragonolobus, Tetragonolobus
Lotus, LTL
purprea( Winged Pea, Asparagus Pea) seeds
LSL-N
E. coli expressed Laetiporus sulphureus
MAA, MAL, MAL-I Maackia amurensis seeds
MalectinE. coli expressed human malectin
MNA-G
Black mulberry
MNA-M
Black mulberry
MOA
E. coli expressed Marasmius oreades
MPL, MPA
Maclura pomifera (Osage Orange) seeds
NPA, NPL, DL
Narcissus pseudonarcissus (Daffodil) bulbs
OrysataE. coli expressed Oryza sative
PA-IIL
E. coli expressed Pseudomonas aeruginosa
PA-IL
E. coli expressed Pseudomonas aeruginosa
PALa
E. coli expressed Phlebodium aureum
Phaseolus vulgaris Erythroagglutinin (Red
PHA-E
Kidney Bean) seeds)
Phaseolus vulgaris Erythroagglutinin (RedPHA-L
Kidney Bean) seeds)

LPA

αFuc
LacNAc, poly LacNAc
Galβ4GlcNAc
Glc2-N -biose

Galactose
MannoseGalα1-3[Fucα1-2]Galβ1-4GlcNAc (Blood Group B) Galα13Galβ1-4GlcNAc, Galα1-3Gal

Galβ3GalNAc
αMan
High-mannose

Fucose, Fucose containing oligosaccharides,
Galα1-3(4)Gal
High-mannose
Galβ4GlcNAcβ2Manα6(GlcNAcb4)(GlcNAcβ4Manα3)Manβ4
Galβ4GlcNAcβ6(Gl cNAcβ2Manα3)Manα3
Galβ4GlcNAcβ2Manα6(GlcNAcb4)(GlcNAcβ4Manα3)Manβ4, Galβ4GlcNAcβ6(GlcNAcβ2Manα3)Manα3
Galβ3GalNAc
α/βGalNAc
αMan, αGlc
α2-6 Sialic Acid

71 Psophocarpus
psophocarpus tetragonolobus
72 lectin ii
73 Phytolacca americana lectin

Phaseolus vulgaris Erythroagglutinin (Red
Kidney Bean) seeds)
PNA
Arachis hypogaea Peanut
PPL
E. coli expressed Pleurocybella porrigens
PSA, PEA
Pisum sativum (Pea) seeds
PSL1a
E. coli expressed Polyporus squamosus
PTL, PTL-I, WBA- Psophocarpus tretragonoiobus (WingedIBean) seeds
Psophocarpus tretragonoiobus (Winged
PTL-II, WBA-II
Bean) seeds
PWM, PWA
Phytolacca americana (Pokeweed)

74 Ricinus communis agglutinin ii

RCA 60

Ricinus communis (Castor bean)

Galactose, N-acetyl-D-galactosamine, lactose

75 Ricinus communis agglutinin I

RCA 120

Ricinus communis (Castor bean)

galactose, lactose

66 Phaseolus vulgaris agglutinin
67 Peanut
68 Pleurocybella porrigens lectin
69 Pisum sativum
70 Polyporus squamosus lectin

PHA-P

N-acetylgalactosamine
GalNAc,Gal(βanomericconfiguration), blood group H structures
and the T-antigen
GlcNAc(β1,4) GlcNAc oligomers, [Gal-(β1,4)16
RayBiotech Lectin Array 95 Kit

Lectins
76 Robinia pseudoacacia lectin
77 Ralstonia solanacearum lectin
78 Sambucus Sieboldiana Lectin
79 Soybean
80 Salivia horminum lectin
81 Sophora japonica
82 Sambucus nigra I
83 Sambucus nigra II
84 Salvia sclarea lectin
85 Solanum tuberosum

Abbreviation Source
RPA
black locust
RS-FucE. coli expressed Ralstonia solanacearum
SAMB
Japanese elderberry
SBA
Glycine max (Soybean) seeds
SHA
Salivia horminum
SJA
Sophora japonica (Japanese Pagoda Tree)
SNA-I
Sambucus nigra (Elderberry) bark
SNA-II
Sambucus nigra (Elderberry) bark
SSA
Salvia
STL, PL
Solanum tuberosum, (potato) tubers

86 Tulipa lectin
87 Urtica dioica
88 Ulex europaeus I
89 Ulex europaeus II
90 Vicia faba
91 Vicia villosa
92 Vicia villosa
93 Vigna radiata lectin
94 Wisteria floribunda
95 Wheat Germ

TL
UDA
UEA-I
UEA-II
VFA
VVA, VVL
VVA-M
VRA
WFA
WGA

Carbonhydrate specificity
N-Acetylgalactosamine, thyroglobulin
FucoseNeuAcα2-6Gal/GalNAc
α > βGalNAc
N-Acetylgalactosamine
βGalNAc
NANAα(2,6)GalNAc > GalNAc = Lac >GalNAc > Gal
N-Acetylgalactosamine(GlcNAc) 2-4
Tulipa sp.
N-Acetylgalactosamine
Urtica dioica (Stinging Nettle) seeds
GlcNAcUlex europaeus (Furze Gorse) seeds
αFucUlex europaeus (Furze Gorse) seeds
Poly β(1,4)Gl cNAc
Vicia faba (Fava Bean) seeds
αMan
Vicia villosa (Hairy Vetch) seeds
GalNAcVicia villosa (Hairy Vetch) seeds
Mannosemung bean
a-Galactose
Wisteria floribunda (Japanese Wisteria) seeds GalNAc
Triticum volganis (Wheat Germ)
GlCNAcSugar Abbreviations
Fuc: L-Fucose
GlcNAc: N -Acetylglucosamine

Gal: D-Galactose GalNAc: N -Acetylglactosamine
Lac: Lactose

Glc: D-Glucose

Man: Mannose

VIII. Application 1 – Detection of Glycans on a Purified Protein
In this application, the RayBio Lectin Array 95 was used to detect specificglycosylations of purified Horseradish Peroxidase (HRP). Lectins BANLEC,
BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL, and PALa showed strong signals
after incubation with 3.3 ug/mL Biotin-HRP followed by detection withstreptavidin-fluorescence-dye (Figures A, B and C). The fluorescence signals
from BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL, and PALa were
blocked in a concentration-dependent manner by HRP itself (Figures A andC), indicating that the signals were generated by lectin-HRP binding. Theseeight lectins are known to exhibit specific binding to mannose, whichindicates that HRP contains mannose. After adding increasing amounts ofmannose, the signals from BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PAIIL, and PALa were reduced (Figures A and B). The reduction in signals fromincreasing concentrations of mannose confirms that HRP protein contains
mannose in its glycocalyx. Additionally, the two lectins AAL and RS-FUC(fucose binding specificity) also showed strong interaction with HRP, whichindicates the fucosylation of HRP. Overall, the results of the Lectin Array 95
were consistent with published literature regarding HRP glycosylation.
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RayBiotech Lectin Array 95 Kit

A
AAL, AMA, BANLEC
BC2L-A, CALSEPA, ConA

GNA, HHA, LAL
MNA-M, NPA, PA-IIL
PALa
RS-FUC

Biotin HRP

Biotin HRP +3mM Mannose

Biotin HRP +30mM Mannose

Biotin HRP +300mM Mannose

Biotin HRP +3ug/ml HRP

Biotin HRP +30ug/ml HRP

Biotin HRP +300ug/ml HRP

AAL,AMA, BANLEC
BC2L-A, CALSEPA, ConA

GNA, HHA, LAL
MNA-M, NPA, PA-IIL

PALa
RS-FUC

Biotin HRP

B
12000

C
0

3mM Mannose

30mM Mannose

300mM Mannose

12000

8000

8000

4000

4000

0

0

0
30ug/ml HRP

3ug/ml HRP
300ug/ml HRP

IX. Application 2 – Profiling of a Serum Sample
Using the lectin 95 array, we can discover the different glycoprotein profilesof serum samples, cell lysates, or purified glycoproteins. The images below
show the profiles of the glycans from different types of samples including
18
RayBiotech Lectin Array 95 Kit

human serum, recombinant glycoproteins human HE4 and AFP, mouse TFF2,
purified human IgG, and bacterial DE3 cell lysates detected by Biotin labelingand fluorescent dye-streptavidin.

Human Serum

HE4

AFP

mTFF2

Anti-Human IgG

HRP

DE3

Blank

X. Other Applications:
Quantitative analysis of lectin-glycoprotein interactions, Example: aconcentration series of glycoproteins detected with the lectin array could
reveal concentration dependent effects of lectin-glycan binding.
Determine the profile of bacterial cell-surface glycans, Example: cell lysate
from bacteria can be biotinylated and hybridized to the lectin 95 array.
Analysis of the binding pattern and correlation with the known carbohydratebinding specificities of the lectins can determine the glycans present on thecell membrane.

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RayBiotech Lectin Array 95 Kit

XI. Troubleshooting Guide
Problem

Cause
Inadequate detection
Inadequate reagent volumes or
improper dilution
Short incubation time

Weak Signal
Too low glycan concentration insampleImproper storage of kit
Bubble formed during incubation
UnevenSignalGeneralHigh
Background

Arrays are not completed coveredby reagent
Reagent evaporation
Cross-contamination fromneighboring wells
Comet tail formation
Inadequate detection

Overexposure
Dark spots
Insufficient wash
Dust
Slide is allowed to dry out

Recommendation
Increase laser power and PMT parameters
Check pipettes and ensure correct preparation
Ensure sufficient incubation time or change sample
incubation step to overnight
Reduce amount of dilution or concentrate sample
Store kit as suggested temperature; Don’t
freeze/thaw the slide
Handle and pipette solutions more gently; De-gas
solutions prior to use
Prepare more reagent and completely cover arrayswith solution
Cover the incubation chamber with adhesive film
during incubation
Avoid overflowing wash buffer
Air dry the slide for at least 1 hour before usage
Increase laser power that the highest concentrationfor each lectin receives the highest possible reading
yet remains unsaturated
Lower the laser power
Completely remove wash buffer in each wash step
Increase wash time and use more wash buffer
Minimize dust in work environment before starting
experiment
Take additional precautions to prevent slides fromdying out during experiment

Note:
This product is intended for research only and is not to be used for clinicaldiagnosis. Our products may not be resold, modified for resale, or used to
manufacture commercial products without written approval by RayBiotech,
Inc.

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RayBiotech Lectin Array 95 Kit

Under no circumstances shall RayBiotech be liable for any damages arisingout of the use of the materials.
Products are guaranteed for six months from the date of purchase whenhandled and stored properly. In the event of any defect in quality or
merchantability, RayBiotech’s liability to buyer for any claim relating to
products shall be limited to replacement or refund of the purchase price.

This product is for research use only.

©2010 RayBiotech, Inc.

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RayBiotech Lectin Array 95 Kit