RayBiotech Lectin Array 70--Detect glycan profiles using 70 lectins

User Manual (Version 3)
October 4, 2021

Cat # GA-Lectin-70

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 (70)AAA, AAL, ACG, ACL, ASA, BanLec, BC2L-A, BC2LCN, BPA,
Calsepa, CGL2, CNL, Con A, DBA, Discoidin I, Discoidin II,
DSA, ECA, EEL, F17AG, Gal1, Gal1-S, Gal2, Gal3, Gal3C-S,
Gal7-S, Gal9, GNA, GRFT, GS-I, GS-II, HHA, Jacalin, LBA, LCA,
LEA, Lentil, Lotus, LSL-N, MAA, Malectin, MOA, MPL, NPA,
Orysata, PA-IIL, PA-IL, PALa, PHA-E, PHA-L, PHA-P, PNA, PPL,
PSA, PSL1a, PTL, RS-Fuc, SAMB, SBA, SJA, SNA-I, SNA-II, STL,
UDA, UEA-I, UEA-II, VFA, VVA, WFA, WGA

FormatOne standard glass slide is spotted with 14 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 70 Kit

TABLE OF CONTENTS
I.

Overview………………………………………………………………………………………………………………………………..………………….… 1
Introduction…................................................................................................................................................3
How It Works………………………………………………………………………………………………………………………………………... 4II.

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

III.

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

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

V.

Lectin Array 70 Map...……………………………………………………………….………………………………………………… 14

VI.

Lectin Array 70 Key……………………………………………………………………………………………………………….

14

VII. Application 1- Detection of Glycans on HRP…………………………………..………...16
VIII. Application 2- Profile of Serum Sample……………………………………………….……..….. 17
IX.

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

X.

Troubleshooting Guide…………………………………………………………………………………………………………....18
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RayBiotech Lectin Array 70 Kit

I.

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 glycan arrays to help researchers: 1) identify and profile theglycans in their samples; 2) determine whether their biomarker of interest
has glycan moieties, and; 3) find specific glycan binding ligands in biologicalsamples.
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.
For the RayBiotech lectin array, one standard glass slide is spotted with 14
identical lectin arrays, 1 in each well. Each lectin, together with the positive
controls is arrayed in duplicate. The slide comes with a 16-well removablegasket which allows for the processing of 14 samples using one slide. Fourslides can be nested into a tray, which matches a standard microplate andallows for automated robotic high-throughput processing of 56 arrays
simultaneously. The RayBiotech lectin array provides a powerful new tool for
glycosylation determination, drug discovery and biomarker development; allwith limited sample volumes required.

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RayBiotech Lectin Array 70 Kit

How It Works

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RayBiotech Lectin Array 70 Kit

II.

Materials Provided

Upon receipt, all components of the RayBiotech Lectin Array 70 kit should be
stored at -200C. 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
28 vials/2
racks
4
1
1
1
1
1
1

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

4-Slide kit
112 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 70 Kit

III. General Considerations
A. Label-Based vs. Sandwich-Based Method
The RayBiotech Lectin Array 70 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 thismanual. 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.
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RayBiotech Lectin Array 70 Kit

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.
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.

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RayBiotech Lectin Array 70 Kit

IV. 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-folddilution). 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.
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.
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RayBiotech Lectin Array 70 Kit

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.
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.
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RayBiotech Lectin Array 70 Kit

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. 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 cell

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RayBiotech Lectin Array 70 Kit

and 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 andframe 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
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RayBiotech Lectin Array 70 Kit

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

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RayBiotech Lectin Array 70 Kit

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
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."13
RayBiotech Lectin Array 70 Kit

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"V.

Lectin Array 70 Map

VI. Lectin Array 70 Key
Lectins

Abbreviation

SourceCarbonhydrate specificity

1 Anguilla anguilla
2 Aleuria aurantia
3 Agrocybe cylindracea lectin
4 Amaranthus caudatus
5 Allium sativum

AAA

Anguilla anguilla (Fresh Water Eel)
Aleuria aurantia mushrooms
E. coli expressed Agrocybe cylindracea galectin
Amaranthus caudatus seeds
Allium sativum agglutinin (Garlic)

6 Musa acuminata lectin
7 Burkholderia cenocepacia lectin

BanLecαFuc
Fucα6GlcNAc
α2-3 Sialic Acid
Galβ3GalNAc
αMan
Mannose, Glucose, branched high-mannose
containing α1,3-glycoside bondHigh-mannose

8 Burkholderia cenocepacia lectin
9 Bauhinia purpurea
10 Calystegia sepium lectin

BC2LCN（AiLecS1)
BPA, BLP
Calsepa

E. coli expressed Burkholderia cenocepacia
Bauhinia purpurea alba (Camel's Foot Tree)E. coli expressed Calystegia sepium

11 Coprinopsis cinerea lectin

CGL2

E. coli expressed Coprinopsis cinerea

12 Clitocybe nebularis lectin
13 Coanavalin A

CNL

E. coli expressed Clitocybe nebularis
Coanavalia ensformis (Jack Beans) seeds

14 Dolichos biflorus
15 Dictyostelium discoideum lectin

DBA

16 Dictyostelium discoideum lectin
17 Datura stramonium
18 Erythrina cristagalli
19 Eunonymus europaeus
20 E. coli lectin
21 Human galectin1 lectin (stable form)

Discoidin II

AAL

ACG
ACL, ACA
ASA

BC2L-A

Con A

Discoidin I

DSA, DSL
ECA, ECL
EEL
F17AG
Gal1

RayBiotech Lectin Array 70 Kit

E. coli expressed Musa acuminata
E. coli expressed Burkholderia cenocepacia

Fucα1-2Galβ1-3GalNAc (H type 3), Fucα1-2Galβ13GlcNAc (H type 1)Galβ3GalNAc
High-mannose
βGal, GalNAcα1-3Gal (Blood Group A), Galα13Gal (Blood Group B)
α/βGalNAc, GalNAcβ1-4GlcNAc, GalNAcα1-3[Fucα12]Galβ1-4GlcNAc (Blood Group A)

αMan, αGlc

Dolichos biflorus (Horse Gram) seeds
E. coli expressed Dictyostelium discoideum

αGalNAc (Tn antigen), LacNAc

αGalNAc

DaturaE.
coli expressed
stramonium
Dictyostelium(Thorn Apple,
discoideum
Jimson Weed)
seeds
Erythrina cristagalli (Coral Tree) seeds
Eunonymus europaeus (Spindle Tree) seeds
E. coli expressed E. coli
E. coli expressed human galectin1 (stable form)

Gal, LacNAc, Asialoglycans, Gal/GalNAcβ14GlcNAcβ1-6Gal/GalNAc(GlcNAc)2-4
Galβ4GlcNAc
Galα3Gal
GlcNAcbranched LacNAc, Gal

14

Lectins
22 Human galectin1-S lectin
23 Human galectin2 lectin
24 Human galectin3 lectin (full-length)
25 Human galectin 3C-S lectin
26 Human galectin7-S lectin
27 Human galectin9 lectin (Stable Form)
28 Galanthus nivalis
29 Griffithia sp. Lectin
30 Griffonia (Banderaea) simplicifolia I
31 Griffonia (Brandeiraea)
32 Hippeastrum hybrid
33 Jacalin
34 Phaseolus lunatus
35 Lens Culinaris
36 Lycopersicon esculentum
37 Lentil lectin

Abbreviation

SourceE. coli expressed human galectin1-S

Gal1-S

Gal2
Gal3
Gal3C-S

Gal7-S
Gal9
GNA, GNL
GRFT
GS-I, GSL-II, BSL-I
GS-II, GSL-II, BSL-II
HHA, HHL, AL
Jacalin, AIL
LBA
LcH, LCA
LEA, LEL, TL
Lentil38 Lotus tetragonolobus
39 Laetiporus sulphureus lectin
40 Maackia amurensis I
41 Human malectin lectin

Lotus, LTL

42 Marasmius oreades lectin
43 Maclura pomifera
44 Narcissus pseudonarcissus
45 Oryza sative lectin

MOA

46 Pseudomonas aeruginosa lectin
47 Pseudomonas aeruginosa lectin
48 Phlebodium aureum lectin

PA-IIL49 Phaseolus vulgaris Erythroagglutinin

PHA-E

50 Leucoagglutinin

PHA-L

51 Phaseolus vulgaris agglutinin
52 Peanut
53 Pleurocybella porrigens lectin
54 Pisum sativum
55 Polyporus squamosus lectin
56 Psophocarpus
57 Ralstonia solanacearum lectin
58 Sambucus Sieboldiana Lectin
59 Soybean
60 Sophora japonica

PHA-P

61 Sambucus nigra I
62 Sambucus nigra II
63 Solanum tuberosum
64 Urtica dioica
65 Ulex europaeus I
66 Ulex europaeus II
67 Vicia faba
68 Vicia villosa
69 Wisteria floribunda
70 Wheat Germ

SNA-I

LSL-N
MAA, MAL, MAL-I
MalectinE. coli expressed human galectin2
E. coli expressed Human galectin3(full-length)
E. coli expressed Human galectin 3C-S
E. coli expressed Human galectin7-S
E. coli expressed human galectin9
Galanthus nivalis (Snowdrop) bulbs
E. coli expressed Griffithia sp.
Griffonia (Banderaea) simplicifolia seeds
Griffonia (Banderaea) simplicifolia seeds
Hippeastrum hybrid (Amaryllis) bulbs
Artocarpus integrifolia (Jackfruit) seeds
Phaseolus lunatus (Lima Bean) seeds
Lens culinaris (lenti I) seeds
Lycopersicon esculentum (tomato) fruit
Lens culinaris seeds
Lotus tetragonolobus, Tetragonolobus
purprea( Winged Pea, Asparagus Pea) seeds
E. coli expressed Laetiporus sulphureus
Maackia amurensis seeds
E. coli expressed human malectin
E. coli expressed Marasmius oreades
Maclura pomifera (Osage Orange) seeds
Narcissus pseudonarcissus (Daffodil) bulbs
E. coli expressed Oryza sative

MPL, MPA
NPA, NPL, DL
Orysata

PALa

PNA
PPL
PSA, PEA
PSL1a
PTL, PTL-I, WBA-I
RS-Fuc
SAMB
SBA
SJA

SNA-II
STL, PL
UDA
UEA-I
UEA-II
VFA
VVA, VVL
WFA
WGA

αFuc
LacNAc, poly LacNAc
Galβ4GlcNAc
Glc 2-N -biose
Galα1-3[Fucα1-2]Galβ1-4GlcNAc (Blood Group B)
Galα1-3Galβ1-4GlcNAc, Galα1-3Gal

Galβ3GalNAc
αMan
High-mannose

E. coli expressed Pseudomonas aeruginosaE. coli expressed Pseudomonas aeruginosaE. coli expressed Phlebodium aureum
Phaseolus vulgaris Erythroagglutinin (Red Kidney
Bean) seeds)
Phaseolus vulgaris Erythroagglutinin (Red Kidney
Bean) seeds)

PA-IL

Carbonhydrate specificity
branched LacNAc
GalNAcα1-3Gal (Blood Group A), branched
LacNAcpoly LacNAc
poly LacNAc
Galβ1-3GlcNAc
poly LacNAc, GalNAcα1-3Gal (Blood Group A)
αMan
High-mannose
αGal, α3GalNAc
α or βGlcNAc
αMan
Galβ3GalNAc
GalNAcα(1,3)[αFuc(1,2]Gal
αMan, αGlc
(GlcNAc)2-4
D-Mannose, D-glucose

Fucose, Fucose containing oligosaccharides,
MannoseGalα1-3(4)Gal
High-mannose
Galβ4GlcNAcβ2Manα6(GlcNAcb4)(GlcNAcβ4Manα3)Manβ4

Galβ4GlcNAcβ6(Gl cNAcβ2Manα3)Manα3
Galβ4GlcNAcβ2Manα6(GlcNAcb4)
Phaseolus vulgaris Erythroagglutinin (Red Kidney (GlcNAcβ4Manα3)Manβ4, Galβ4GlcNAcβ6(GlBean) seeds)
cNAcβ2Manα3)Manα3Arachis hypogaea Peanut
Galβ3GalNAc
E. coli expressed Pleurocybella porrigens
α/βGalNAc
Pisum sativum (Pea) seeds
αMan, αGlc
E. coli expressed Polyporus squamosus
α2-6 Sialic Acid
Psophocarpus tretragonoiobus (Winged Bean)
GalNAc, Gal
E. coli expressed Ralstonia solanacearum
FucoseJapanese elderberry
NeuAcα2-6Gal/GalNAc
Glycine max (Soybean) seeds
α > βGalNAc
Sophora japonica (Japanese Pagoda Tree) seeds βGalNAc
NANAα(2,6)GalNAc > GalNAc = Lac >Sambucus nigra (Elderberry) bark
GalNANAα(2,6)Gal
Sambucus nigra (Elderberry) bark
GalNAc > Gal
Solanum tuberosum, (potato) tubers(GlcNAc)2-4
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
GalNAcWisteria floribunda (Japanese Wisteria) seeds
GalNAcTriticum volganis (Wheat Germ)
GlCNAcSugar Abbreviations
Fuc: L-Fucose
GlcNAc: N -Acetylglucosamine

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

Glc: D-Glucose

Man: Mannose

15
RayBiotech Lectin Array 70 Kit

VII. Application 1 – Detection of Glycans on a Purified Protein
In this application, the RayBio Lectin Array 70 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 signal from BANLEC, BC2L-A, CALSEPA, GNA, HHA, NPA, PA-IIL,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 70
were consistent with published literature regarding HRP glycosylation.
AAL
BANLEC, BC2L-A
CALSEPA
GNA, HHANPA
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
BANLEC, BC2L-A
CALSEPA
GNA, HHANPA
RS-FUC

Biotin HRP

16
RayBiotech Lectin Array 70 Kit

C

B
20000

0ug/ml HRP

3ug/ml HRP

30ug/ml HRP

300ug/ml HRP

30000

0 uM Mannose

3 mM Mannose

30 mM Mannose

300 mM Mannose

15000

20000
10000
10000
5000
0

0

VIII.

Application 2 – Profiling of a

Serum Sample
Using the lectin array, we can discover the different glycoprotein profiles ofthe serum samples, cell lysates, or purified glycoprotein. The images below
show the profiles of the glycans from different types of samples including
human serum, recombinant glycoproteins human HE4, AFP, mouse TTF,
purified human IgG, and bacterial cell lysates OF DH-5α, DE3 detected by
Biotin labeling and Fluorescence dye-streptavidin.

Human SerumHE
4

Anti-Human IgG DH5-alpha

AFP

DE
3

mTTF

Blank
17

RayBiotech Lectin Array 70 Kit

IX. 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; Cell lysate frombacteria can be biotinylated and hybridized to the lectin array. Analysis of thebinding pattern and correlation with the known carbohydrate-binding
specificities of the lectins can determine the glycans on the cell membrane.

X.

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

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
18

RayBiotech Lectin Array 70 Kit

Slide is allowed to dry out

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.
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.

19
RayBiotech Lectin Array 70 Kit