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PROTEOGLYCANS

GLYCOSAMINOGLYCANS

&

Antibodies, Assays, & Enzymes

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http://www.halsanet.com/15/2014/04/lindriga-symtom-ms.html

tableOF CONTENTS

ANTIBODIES List of antibodies 3 Structure of Glycosaminoglycans 4 Specficity of antibodies 6 Application of antibodies 10 Experimental Procedure for Antibodies 18 References for Antibodies 20

RELATED PRODUCTS Hyaluronic Acid Binding Protein 22 GAG Assays 24 Sulfated GAG Quantitation Kit Hyaluronan Assay

Heparan Sulfate ELISA kit Highly Sensitive Keratan Sulfate ELISA Substrates & Standards 28 GAG Arrays & Analysis of Glycosaminoglycan Structure 28

Polysaccharides 28 GAGS, K5 Polysaccharides, Heparins Selectively De-Sulfated Heparins

Oligosaccharides 30 Dermatan Sulfate, Heparin, Hyaluronic Acid Disaccharides 31 Sets and Mixtures, Chondroitin / Dermatan, Heparin Select-HA™, Oligo-HA™ & Nano-HA™ Standards & Ladders 33 Heparin/GAG Binding Plates / GAG Arrays 37 Enzyme Activity Assays 38 Heparanase & Hyaluronidase Assays GAG-coated 96-well plates GAG Degrading Enzymes 39 Purified & recombinant enzymes (including new K5 Heparan Lyase) Alternative Names for GAG-degrading enzymes Enzymes / Substrates Overview Select-HA, Oligo-HA and Nano-HA are trademarks of Hyalose LLC

oneLIST OF ANTIBODIES

1

��

Ventral membranes of gizzard

fibroblast CS-56 Chondroitin sulfate IgM �� 0.5mL

Proteoglycan from chick embryo

limb bud MO-225 Chondroitin sulfate Type D IgM �� 0.2mg

Adult rat bone protein MC21C Chondroitin-6-sulfate IgM �� 0.2mg

Chicken type IX collagen containing

chondroitin-4-sulfate LY111 Chondroitin-4-sulfate IgM �� 0.2mg

CSPG digested with chondroitinase

ABC 1-B-5 Di-0S IgG1 �� 0.2mg

Same as above 2-B-6 Di-4S IgG1 �� 0.2mg

Same as above 3-B-3 Di-6S IgM �� 0.2mg

PG from 10-day-old rat brain 2H6 Chondroitin Sulfate A IgM �� 0.2mg

Extracts of monkey brain 473A12 Chondroitin IgA �� 0.2mg

Capsular polysaccharides

from E. coli K5

Heparan sulfate proteoglycan from

rat glomerular basement membrane JM403 Heparan sulfate IgM �� 0.2mg

Mouse fibrosarcoma induced by

Meth-A HepSS-1 Heparan sulfate IgM �� 0.2mg

HSPG from human fetal lung

fibroblast F58-10E4 Heparan sulfate IgM �� 0.2mg

HSPG from human fetal lung

fibroblast digested with heparitinase F69-3G10 Heparan sulfate IgG2b �� 0.2mg

Chondroitin sulfate family

Heparan sulfate family

NAH46 Heparan sulfate IgM �� 0.2mg

CSPG monomer from human

articular cartilage digested with

chondroitinase ABC 5-D-4 Keratan sulfate IgG1 �� 0.2mg

A proteoglycan core antigen

prepared by chondroitinase ABC

digestion of human adult cartilage

proteoglycan monomer BCD-4 Keratan sulfate IgG1 �� 0.2mg

Dermatan sulfate proteoglycan

(Decorin)

Human yolk sac tumor 2-B-1 Large proteoglycan (Versican) IgG1 �� 0.2mg

HSPG from mouse EHS tumor HK-102 HS proteoglycan (Perlecan) Rat IgG2a �� 0.2mg

CSPG from 10-day-old rat brain 1G2 CS proteoglycan (Neurocan) IgG1 �� 0.2mg

PG from 10-day-old rat brain 6B4 CS proteoglycan (Phosphacan) IgM �� 0.2mg

Membrane-bound CSPG purified

from 10-day-old rat brains C1 Neuroglycan C IgM �� 0.2mg

Keratan sulfate

Core protein of proteoglycan

6-B-6Human ovarian fibroma IgG1 �� 0.2mg

∆

∆∆

∆

twoSTRUCTURE OF GLYCOSAMINOGLYCANSHyaluronanChondroitin Sulfate

DermatanSulfate

HeparanSulfate

Heparin

KeratanSulfate

GlcNAcsulfate β linkage α linkage

GalNAc Gal GlcA IdoA4

46 6

3 3 32 22(N)1 1 1

Chondroitin Sulfate FamilyCOOH

H.OHCH2OR1R2OOH O

OO

NHAcOR3

AbbreviationR1 R3R2H

CAD(B)E

-OS∆Di

-6S-4S-US-(U,6)S-(U,4)S-(4,6)S-(U,4,6)S

H HHH

HHH

SO3-

SO3-

SO3-SO3-SO3-

SO3-SO3-SO3-

SO3-

SO3-

SO3-SO3-

HH

HH

Heparan Sulfate FamilyCOOH

H.OHCH2OR1

OR3 NHR2OH O O OOH

R1 R3R2HH

-OS∆DiHS

-NS-6S-US-(6,N)S-(U,N)S-(U,N,6)S

Ac HHH

SO3-

Ac

SO3-

SO3-H

SO3-SO3-

SO3-

AcSO3- SO3-

SO3-

SO3-

HH

Structure of the Unsaturated Disaccharides

2

STRUCTURE OF GLYCOSAMINOGLYCANS

bovine aortabovine lung

bovine lungpig intestine

OS NS 6S US (6,N)S (U,6,N)S(U,N)S

OS NS 6S US (6,N)S (U,6,N)S(U,N)SHEPARAN SULFATE*

HEPARIN

bovine intestinebovine kidney (1.1M)

bovine kidney (1.25M)

* Maccarna, M. et al., J. Biol. Chem., 271, 17804-17810 (1996)

sturgeon notochordwhale cartilageshark cartilage

squid cartilageshark fin

bovine kidneypig skin

CHRONDROITIN SULFATE

DERMATAN SULFATE

OS 6S 4S (U,6)S (U,4)S (U,4,6)S(4,6)S

OS 6S 4S (U,6)S (U,4)S (U,4,6)S(4,6)S

Disaccharide Composition of Chondroitin Sulfate and Dermatan Sulfate

Disaccharide Composition of Heparan Sulfate and Heparin

3

threeHRP labeled anti-mouse antibody

Anti-GAG monoclonal antibodies

Biotinylated GAGs

Avidin

Microplate

TMB (substrate) Measure the absorbance at 450nmHRP

SPECIFICITY OF ANTIBODIES

HRP labeled anti-mouse antibody

Anti-∆ GAG monoclonal antibodies

Biotinylated GAGs or PGs

in the case of 3G10, 1-B-5, 2-B-6, 3-B-3

Enzymes

Inhibitors

Microplate

TMB (substrate) Measure the absorbance at 450nmHRP

Assay Method for GAG Antibodies

Assay Method for ∆GAG Antibodies

4


MO-225

A450

CS-D

CS-ECS-C

CS-A (whale)

0.001 0.01 0.1 1

3.02.52.01.51.00.50.0

(0.5)

CS-56

A450

CS-D

3.02.52.01.51.00.50.0

(0.5)

LY111

Biotinylated GAGs (µg/ml)

CS-DCS-A

A450

3.02.52.01.51.00.50.0

(0.5)

CS-DCS-E CS-C

3.02.52.01.51.00.50.0

(0.5)

MC21C

Ch

4733.02.52.01.51.00.50.0

(0.5)


CS-A

CS-D

2H63.02.52.01.51.00.50.0

(0.5)

HAChCS-C (shark) CS-B (pig)CS-A (whale) CS-A (sturgeon)CS-D (shark) CS-E (squid)Heparin (pig) HS (bovine)KS (bovine)

0.001 0.01 0.1 1

0.001 0.01 0.1 1

0.001 0.01 0.1 100101

0.1 100101

0.01 0.1 1 10

Chondroitin SulfateReactivity of Antibodies to Glycosaminoglycans

5

3.02.52.01.51.00.50.0 0.0001 0.01 1 100

EHS-HSHep (pig)

HepSS-13.02.52.01.51.00.50.0

10E4EHS-HS

0.0001 0.01 1 100

Hep (pig)HS (bovine)A45

0

3.02.52.01.51.00.50.00.0001 0.01 1 100

Hep (pig)EHS-HS

CS-E

HK249


A450

3.02.52.01.51.00.50.0

0.1 10 100 1000

5D4


A450

1 10 100 1000

3.02.52.01.51.00.50.0

HABP HA

CS-A (sturgeon)CS-D (shark)KS (bovine)

CS-A (whale)CS-C (shark)HS (bovine)

Heparin (pig)CS-B (pig)CS-E (squid)HA

CH

EHS-HSCS-A (whale)CS-C (shark)

Hep (pig)CS-B (pig)KS (bovine)

HS (bovine)CS-A (sturgeon)CS-E (squid)

HACS-D (shark)CH

SPECIFICITY OF ANTIBODIESHeparan Sulfate

Hyaluronic Acid & Keratan Sulfate

Reactivity of Antibodies to Glycosaminoglycans

5D4

6

∆Di-CS 0S∆Di-HS NS∆Di-HS diS(NS, 6S)∆Di-HS 6S∆Di-HS diS(US, NS)∆Di-HS triS(NS, 6S, US)

Inhibitio

n (%)

10090807060504030201000.01 1.00 100.00 1000.00

Disaccharide Concentration (nmol/ml)


3-B-3

Inhibitio

n (%)

100908070605040302010

0

∆Di-CS 6S

∆Di-CS 0S

0.01 0.1 1 10Diasaccharide concentration (µg/ml)

1009080706050403020100

1-B-5∆Di-CS 0S

0.01 0.1 1 10

Inhibitio

n (%)

2-B-6

Inhibitio

n (%)

0.01 0.1 1 10

∆Di-CS 4S1009080706050403020100

∆Di-CS 0S∆Di-CS diSE∆Di-CS diSD∆Di-CS 4S∆Di-CS 6S∆Di-CS triS

∆ Chondroitin Sulfate

∆ Heparan Sulfate

Reactivity of Antibodies to Unsaturated Glycosaminoglycans

3G10∆Di-HS diS (NS, 6S)

∆Di-HS 6S

∆Di-HS NS∆Di-HS triS (NS, 6S, US)

7

fourAPPLICATION OF ANTIBODIESImmunohistochemistry of Articular Cartilage using MAb to Proteoglycan ∆Di-0S, ∆Di-4S & ∆Di-6S

Figure 1: Chase ABC-

Figure 2: Chase ABC+ Surface Layer Middle Layer Deep Layer





∆Di-4S(Clone 2-B-6)



8

APPLICATION OF ANTIBODIESImmunostaining using MAb (10E4) to Heparan Sulfate

Immunostaining using MAb (LY111) to Chondroitin Sulfate1) Epidermis 2) Plexus Chorioideus 3) Peripheral Nerve 4) Submaxillary Gland 5) Intestine 6) Kidney

1) Mouth 2) Vertebra 3) Intestine 9

APPLICATION OF ANTIBODIESImmunohistochemistry of Hamster’s Embryo Tissues Using MAb (10E4, 3G10) to Heparan Sulfate.

1) Epidermis 2) Plexus Chorioideus 3) Peripheral Nerve

10E4 HSase (+) 10E4 HSase (+) 10E4 HSase (+)

10E4 HSase (-) 10E4 HSase (-) 10E4 HSase (-)

3G10 HSase (+)

3G10 HSase (-)

3G10 HSase (+) 3G10 HSase (+)

3G10 HSase (-) 3G10 HSase (-)

Hamster Fetus (14 days)

10

APPLICATION OF ANTIBODIES

4) Submaxillary Gland 5) Intestine 6) Kidney

10E4 HSase (+) 10E4 HSase (+) 10E4 HSase (+)

10E4 HSase (-) 10E4 HSase (-) 10E4 HSase (-)

3G10 HSase (+)

3G10 HSase (-)

3G10 HSase (+) 3G10 HSase (+)

3G10 HSase (-) 3G10 HSase (-)11

Immunohistochemistry of Hamster’s Embryo Tissues Using MAb (1-B-5, 2-B-6,3-B-3, LY111, CS-56) to Chondroitin SulfateAPPLICATION OF ANTIBODIES

1) Mouth 2) Vertebra

1-B-5 1-B-5 1-B-5

2-B-6 2-B-6 2-B-6

3-B-3 3-B-3 3-B-3

LY111 LY111 LY111

CS-56 CS-56 CS-56

Chase ABC (-) Chase ABC (+)Chase ABC (+)

Hamster Fetus (14 days)

12

APPLICATION OF ANTIBODIES3) Intestine

1-B-5

2-B-6

3-B-3

CS-56

LY111

1-B-5

2-B-6

3-B-3

CS-56

LY111

1-B-5

2-B-6

3-B-3

CS-56

LY111

Chase ABC (+) Chase ABC (-)Chase ABC (-)

13

APPLICATION OF ANTIBODIESWestern Blot

Western blot analysis of rat liver proteoglycans using MAb to ∆-heparan sulfate (3G10)

Fibroglycan (dimer)Syndecan, Ryudocan(dimer)

Fibroglycan Ryudocan

Front

44kDa

87kDa

144kDa208kDa

HSase I (+)

ImmunoprecipitationImmunoprecipitation analysis of rat liver proteoglycans using MAb to ∆Di-0S (1-B-5), ∆Di-4S (2-B-6)

160 kDa80 kDa

49.5 kDa

32.5 kDa29.5 kDa

Origin

Front

1-B-5 2-B-6A B(-) (+) C D(-) (+)Chase

14

APPLICATION OF ANTIBODIESHSase I (-) HSase I (+)

Cell Line 10E4 3G10 10E4 3G10KM3 Common ALL weakly + 10% ~± → →Daudi Burkitt Lymphoma -~± - → →EB2 Burkitt Lymphoma (ovary) + (>90%) + (60%) ↓ (50%) ↑ (>90%)CCRF-SB ALL (B) weakly + (50%) - ↓ (-) ↑ (70%)Molt 4 ALL (T) - - → →HPBALL ALL (T) weakly + (20%) - ↓ (10%) ↑ (90%)K-562 Erythroleukemia + (80%) - ↓ (10%) ↑ (90%)PB (M) Normal Human Monocyte - - → →PB (L) Normal Human Lymphocyte - - → →PB (G) Normal Human Granulocyte ~± ~± → →MKN 74 Stomach Cancer + (100%) - ↓ (>90%) ↑ (>90%)COLO 201 Colon Cancer + (80%) - ↓ (-) ↑ (100%)PLC/PRF/5 Hepatoma + (80%) ~± ↓ (-) ↑ (50~60%)Hep G2 Hepatocellular Carcinoma + (100%) - ↓ (80%) ↑ (100%)G32TG Hepatocellular Carcinoma + (90%) - ↓ (±) →

Flow Cytometry Using MAb 10E4 and 3G10

K-56210E4 3G10

3G1010E4PLC/PRF/5

+: positive, -: negative, ↑: increase, ↓: decrease, →: no change, (%): positive rate

15

fiveEXPERIMENTAL PROCEDURESReagents Antibodies to Heparan Sulfate: 10E4, 3G10

Antibodies to Chondroitin Sulfate:LY111, CS-56, 1-B-5, 2-B-6, 3-B-3GAGases:Heparitinase I (20 mU/ml of sodium acetate buffer-3.3 mM calcium chloride, pH 7.0)Chondroitinase ABC Protease free (1-5 U/ml of 20 mM Tris-HCI buffer, pH 8.0)

Immunohistochemistry for Heparan Sulfate and Chondroitin Sulfate

References Antibodies to Heparan Sulfate1. David, G. et al. J. Cell Biol., 119, 961-975 (1992)Antibodies to Chondroitin Sulfate1. Mark, M.P. et al. Develop. Biol., 133, 475-488 (1989)2. Fukatsu, T. et al. Br. J. Cancer, 57, 74-78 (1987)3. Sorrell, J.M. et al. J. Immunol., 140, 4263-4270 (1988)

Procedure 1. Prepare slides and controls.2. Preincubate sections with reaction buffer of GAGase for 15 minutes at 37oC.3. Incubate with GAGase for 1-2 hours at 37oC. Wash.4. Block endogenous peroxidase with 0.3% H2O2 methanol.5. Incubate with 1% BSA in PBS for 1 hour at room temperature.6. Incubate with anti-HS or anti-CS antibody for 1-2 hours at room temperature. Wash.7. Incubate with HRP conjugated anti-mouse IgG or IgM for 1 hour at room temperature. Wash.8. Incubate with HRP substrate. Wash.9. Observe by microscopy.

Reagents Antibody to Heparan Sulfate:10E4, 3G10Cells:K-562, PLC/PRF/5GAGases:Heparitinase I (50 mU/ml of phosphate buffer saline, pH 7.4 (PBS)

Procedure 1. Incubate 1 x 106 cells with 100 µl of Heparitinase I or PBS for 20 minutes at 37oC. Wash.2. Incubate cells with anti-HS for 30 minutes at 4oC. Wash.3. Incubate cells with FITC conjugated F(ab’)2 fragment anti-mouse IgG or IgM for 30 minutes at 4oC. Wash.4. Analyze using manufacturers instructions.

Flow Cytometry for Heparan Sulfate

16

EXPERIMENTAL PROCEDURESReagents Antibodies to Chondroitin Sulfate:1-B-5, 2-B-6

GAGases:Chondroitinase ABC Protease free (1-5 U/ml of 100 mM sodium acetate buffer, pH 8.0 or 20 mM Tris-HCI buffer,pH 8.0)

Immunoprecipitation for Chondroitin Sulfate Proteoglycan

Procedure 1. Prepare 125I proteoglycan fractions by Chloramin T method.2. Incubate 125I labeled proteoglycan fractions with GAGase for 1 hour at 37oC.(Treat 3 µg of sample with 100 mU of Chondroitinase ABC)3. Incubate sample with normal mouse IgG and Protein G Sepharose for 1 hour at 4oC.4. Remove Protein G Sepharose binding non-specific immune complexes and save supernatant.5. Incubate supernatant with anti-CS antibody and new Protein G Sepharose for 3 hours at 4oC. Wash.6. Boil Protein G Sepharose binding specific immune complexes with SDS-PAGE sample buffer for 5 minutes.7. Save supernatant after centrifugation.8. Run supernatant on SDS-PAGE under reducing conditions.9. Place gel in direct contact with X-ray film and develop using manufacturers instructions.

AbbreviationsBSA - Bovine serum albumin PBS - Phosphate buffered salineFITC - Fluorescein isothiocyanate HUVECs - human umbilical vein endothelial cellsSDS - Sodium dodecyl sulfate PAGE - polyacrylamide gel electrophoresis

Reagents Antibody to Heparan Sulfate:3G10

GAGases:Heparitinase I (200 mU/ml of sodium acetate buffer-3.3 mM calcium chloride, pH 7.0)Membranes:PVDF membrane or nitrocellulose membrane

References Antibodies to Heparan Sulfate1. Bai, X.M. et al. J. Histochem. Cytochem., 42, 1043-1054 (1994)Antibodies to Chondroitin Sulfate1. Yada, T. et al. J. Histochem. Cytochem., 44, 969-980 (1996)

Procedure 1. Incubate partially purified proteoglycan fractions with GAGase for 1 hour at 37oC.a) Treat 1.5 µg of sample with 2 mU of Heparitinase I for heparan sulfate proteoglycanb) Treat 1 µg of sample with 100 mU of Chondroitinase ABC for chondroitin sulfate proteoglycan.2. Run samples on SDS-PAGE under reducing conditions.3. Transfer it to membrane.4. Blocking with 10% skim milk in PBS for 30 minutes at 37oC.5. Incubate with anti-HS or anti-CS antibody for 1 hour at room temperature. Wash.6. Incubate with HRP conjugated anti-mouse IgG or IgM for 1 hour at room temperature. Wash.7. Incubate with HRP substrate. Wash.

Western Blot for Heparan Sulfate Proteoglycan

17

sixCS-56; Monoclonal antibody (MAb) to Chondroitin Sulfate1. Avnur, Z. et al.: Spatial interrelationships between proteoglycans and extracellular matrix in cell cultures. Exp. Cell. Res., 158,321-332 (1985)2. Hay, E. D. et al.: Extracellular matrix. J. Cell Biol., 91, 205-223 (1981)3. Aplin, J. D. et al.: Complex carbohydrates of the extracellular matrix structures, interactions and biological roles. Biochem.Biophys. Acta, 694, 375-418 (1982)4. Christner, J. E. et al .: Immunological determinants of proteoglycans. J. Biol. Chem., 255, 7102-7105 (1980)5. Grinnell, F. et al.: Cellular adhesiveness and extracellular substrata. Int. Rev. Cytol., 53, 65-144 (1978)MO-225; Monoclonal antibody (MAb) to Chondroitin Sulfate (type D)1. Yamagata, M. et al.: A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain. J. Biol. Chem., 262, 4146-4152 (1987)2. Yamagata, M. et al.: Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates. J. Biol.Chem., 264, 8012-8018 (1989)MC21C; Monoclonal antibody (MAb) to Chondroitin-6-Sulfate1. Mark, M. P. et al.: Transient expression of a chondroitin sulfate-related epitope during cartilage histomorphogenesis in the axial skeleton of fetal rats. Develop. Biol., 133, 475-488 (1989)2. Mark, M. P. et al.: Chondroitin sulfates in developing mouse tooth germs: an immunohistochemical study with monoclonal antibodies against chondroitin-4 and chondroitin-6 sulfates. Differentiation, 43, 37-50 (1990)3. Mark, M. P. et al.: Regulated changes in chondroitin sulfation during embryogenesis: an immunohistochemical approach. Int. J.Dev. Biol., 34, 191-204 (1990)LY111; Monoclonal antibody (MAb) to Chondroitin-4-Sulfate1. Yada, T. et al.: Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. J. Biol. Chem., 267,9391-9397 (1992)1-B-5; Monoclonal antibody (MAb) to Proteoglycan ∆Di-0S2-B-6; Monoclonal antibody (MAb) to Proteoglycan ∆Di-4S3-B-3; Monoclonal antibody (MAb) to Proteoglycan ∆Di-6S1. Couchman, J. R. et al.: Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature, 307,650-652 (1984)2. Sobue, M. et al.: Immunohistochemical localization of chondroitin and dermatan sulfate proteoglycans in human connective tissues. Connect. Tissue, 19, 117-126 (1987)3. Fukatsu, T. et al.: Immunohistochemical localization of chondroitin and dermatan sulfate proteoglycans in tumor tissues. Br. J.Cancer, 57, 74-78 (1987)4. Sorrell, J. M. et al.: Epitope-specific changes in chondroitin sulfate/dermatan sulfate proteoglycans as markers in the lympopoietic and granulopoietic compartments of developing bursae of fabricius. J. Immunol., 140, 4263-4270 (1988)5. Hagiwara, H. et al.: Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage. Histochemistry, 103, 213-220 (1995)HepSS-1; Monoclonal antibody (MAb) to Heparan Sulfate1. Kure, S. and Yoshie, O.: A syngenic monoclonal antibody to murine Meth-A sarcoma (HepSS-1) recognizes heparan sulfate glycosaminoglycans (HS-GAG): cell density and transformation dependent alteration in cell surface HS-GAG defined by HepSS-1.J. Immunol., 137, 3900-3908 (1986)2. Kure, S. et al.: Metastatic potential of murine B16 melanoma correlates with reduced surface heparan sulfate glycosaminoglycan. Jap. Cancer Res. (Gann), 78, 1238-1245 (1987)

REFERENCES FOR ANTIBODIES

18

REFERENCES FOR ANTIBODIESF58-10E4; Monoclonal antibody (MAb) to Glucosamine N-Sulfate of Heparan SulfateF59-3G10; Monoclonal antibody (MAb) to ∆-Heparan Sulfate1. David, G. et al.:Developmental changes in heparan sulfate expression: in situ detection with MAbs. J. Cell Biol., 119, 961-975 (1992)2. Bai, X. M. et al.: Differential expression of multiple cell-surface heparan sulfate proteoglycans during embryonic tooth development. J. Histochem Cytochem., 42, 1043-1054 (1994)3. Fuxe, K. et al.: On the regional distribution of heparan sulfate proteoglycan immunoreactivity in the rat brain. Brain Res., 636,131-138 (1994)4. Nackaerts, K. et al.: Heparan sulfate proteoglycan expression in human lung-cancer cells. Int. J. Cancer, 74, 335-345 (1997)5-D-4; Monoclonal antibody (MAb) to Keratan Sulfate1. Caterson, B. et al.: Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. J.Biol. Chem., 258, 8848-8854 (1983)2. Mehmet, H. et al.: The antigenic determinants recognized by three monoclonal antibodies to keratan sulfate involve sulfated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series. Eur. J. Biochem., 157, 385-391 (1986)473; Monoclonal antibody (MAb) to Chondroitin1. Watanbe, E. et al.” A monoclonal antibody identifies a novel epitope surrounding a subpopulation of the mammalian central neurons. Neuroscience, 29, 645-657 (1987)2. Fujita, S.C. et al.: A novel member of the family of perineuronal antigens associated with subpopulations of central neurons in the rat. Exp. Brain Res., 88, 345-354 (1992)3. Sano, S. et al.: Differential subsets of CNS neurons express different glycosaminoglycan epitopes on large perineuronal proteoglycans. Brain Res., 630, 65-74 (1993)2H6; Monoclonal antibody (MAb) to Chondroitin Sulfate A1. Yamamoto, Y. et al.: Immunohistochemical localization of chondroitin sulfate in the forestomach of the sheep. Eur. J. Histochem.,39, 265-272 (1995)HK-102; Monoclonal antibody (MAb) to Mouse Heparan Sulfate Proteoglycan1. Kato, M. et al.: Basement membrane proteoglycan in various tissues: characterization using monoclonal antibodies to the engelbreth-holm-swarm mouse tumor low density heparan sulfate proteoglycan. J. Cell Biol., 106, 2203-2210 (1988)2. Snow, A. et al.: The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimers disease. Am. J. Pathol., 133, 456-463 (1988)6-B-6; Monoclonal antibody (MAb) to Human Dermatan Sulfate Proteoglycan1. Sobue, M. et al.: Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan. J. Histochem.Cytochem., 36, 479-485 (1988)2-B-1; Monoclonal antibody (MAb) to Human Large Proteoglycan1. Sobue, M. et al.: Production and characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumor. Histochem. J., 21, 455-459 (1989)2. Isogai, Z. et al.: 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors, is a large chondroitin sulfate proteoglycan, PG-M/Versican. Cancer Res., 56, 3902-3908 (1996)3. Nara, Y. et al.: Immunohistochemical localization of extracellular matrix components in human breast tumors with special reference to PG-M/Versican. Histochem. J., 29, 21-30 (1997)

19

Biotinylated Hyaluronic Acid Binding Protein (B-HABP)

Cat. No. 400763-1A Source Bovine nasal cartilage Purification The hyaluronic acid binding region of the proteoglycan extracted with 4M guanidine HCl

and then by affinity chromatography using HA coupled resin.

Introduction Purified cartilage proteoglycans bind specifically to hyaluronate to form high-MW aggregates in which many proteoglycans are bound to each hyaluronate chain. The proteoglycans bind by a specific site at one end of the protein backbone that is largely devoid of glycosaminoglycan chain(HA-binding region) and has a high affinity for a decasaccharide unit of hyaluronate

A,B). The link protein

C) is an integral part

of the aggregate structure and has been proposed to form additional bonds, by bridging the proteglycan molecule and the hyaluronate chain, thereby increasing the strength of binding and giving a more stable aggregate structure(ternary complex).

D,E) Purification of hyaluronic acid binding protein (HABP) from

bovine nasal cartilage was performed in accordance with the modified method of E Laurent et al.D)

Postulated model of HABP The protein core of the aggrecan which bind to hyaluronic acid consists of region which interacts with hyaluronic acid and a region to which the majority of the polysaccharide chains are covalently attached.

Applications • ELISA

(H-J)

• Histochemistry (K-N)

ELISA Procedure for Hyaluronic Acid using B-HABP

1. Coat Costar plate with HA (1 ug) or use BSA-HA plates 2. Wash with T-PBS buffer (200ul) 3 times 3. Add bHABP (e.g. 50 ng/mL) [T-PBS/1% BSA as a negative control] 4. Incubate covered by Parafilm at 37 C for 1 hour 5. Discard excess bHABP 6. Wash with T-PBS buffer (200ul) 3 times 7. Add 100ul HRP-sAv solution (ImmunoPure® Streptavidin, Horseradish Peroxidase Conjugated,

1mg) 8. Incubate covered by Parafilm at 37 C for 1 hour 9. Discard excess HRP-SAV 10. Wash with T-PBS buffer (200ul) 3 times 11. Add 100 ul TMB substrate 12. Incubate covered by Parafilm at 37 C for 15 min (prevent light exposure) 13. Add 100ul 1N HCl 14. Measure the endpoint at A450 minus A630nm

20

sevenRELATED PRODUCTS

Histochemistry for Hyaluronic acid using B-HABP

Reagents

Product Source Cat.No.

Biotinylated Hyaluronic Acid Binding Protein Seikagaku 400763-1A Hyaluronidase (Streptmyces hyalurolyticus) Seikagaku 100740-1 Chondroitinase ABC Protease Free (Proteus vulgaris) * Seikagaku 100332-1A Trypsin * Sigma Avidin solution/ Biotin solution Vector Fluorophore conjugated Streptavidin Jackson * For proteoglycan digestion when HA is masked.

Experimental procedure

1. Pretreatment with Hyaluronidase (Negative control) Treat with reaction buffer of Hyaluronidase (100mM Sodium acetate buffer, pH6.0) for 15min at 37°C. No wash Treat with Hyaluronidase (200TRU/mL; 100mM Sodium acetate buffer, pH6.0) for 2hrs at 60°C. Wash with PBS 2. Blocking endogeneous avidin biotin activity Treat with avidin solution for 20min at RT. Wash Treat with biotin solution for 20min at RT. Wash 3. Treat with 0.1% BSA solution for 1hr at RT. Wash 4. Treat with Biotinylated HABP (2µg/mL) for 1-2hrs at RT. Wash 5. Treat with Fluorophore conjugated Streptavidin for 15min at RT.

Example: Histochemical staining of hyaluronan (Texas red) in rabbit cornea.

N: Normal cornea; 1, 3, 14 and 28 days: corneas 1, 3, 14 and 28 days after wounding

References for HABP

A. Hardingham,T.E.and Muir,H.Biochim.Biophys.Acta,279,401(1972) B. Hascall,V.C.and Heinegard,D.J.Biol.Chem.,249,4242(1974) C. Heinegard,D.and Hascall,V.C.J.Biol.Chem.,249,4250(1974) D. Hardingham,T.E.Biochem.J.,177,237(1979) E. Kimura,J.H.,Hardingham,T.E.,Hascall,V.C.and Solursh,M.J.Biol.Chem.,254,2600(1979) F. E-Laurent,A.and Hallgren,R.Ann.Rheum.Dis.,44,83-88(1985) G. Laurent T.C.and Fraser JRE.Ciba foundation Symposium 124,9-29(1986) H. E-Laurent A,et al.Hepatology 5,638-642(1985) I. Frebourg T,et al.Hepatology 6,392-395(1986) J. Kondo, K., Chichibu, K., Usuki, H., Matsuura, T., Shichijo, S. and Yokohama, M. : Jpn. J. Clin. Pathol., 39, 536-540(1991) K. Watanabe, N., Kato, S., Hamai, A. and Sakurai, K. : Connect. Tiss., 22, 71-78(1991) L. Toole,B.P.,Munaim,S.I.,Welles,S.and Kundson,C.B.Ciba Foundation Symposium 143,138-149(1989) M. West,D.C.,and Kumar,S.Ciba Foundation Symposium 143,187-207(1989) N. Uzuki, M. and Sawai, t. : Connect. Tiss., 25, 251-262(1994)

21

RELATED PRODUCTS

Code No.：280560

Sulfated Glycosaminoglycan Quantitation Kit

This kit is based on a colorimetric method using 1,-9,-Dimethylmethylene Blue (DMMB) specific to sulfated Glycosaminoglycan (GAG) and is developed for measurement of sulfated GAG in tissue extract, culture medium and culture cells.

Please read this insert completely before using this product.

［Contents of the Kit］

Contents Amount Number

A Microplate 96 well plate 2 plate

B Sulfated GAG Standard (1,000!g/mL) 1 mL 1 vial

C DMMB Dye Solution 34 mL 1 vial

D Reaction Buffer Ⅰ 14 mL 1 vial

E Reaction Buffer Ⅱ 14 mL 3 vial

F Concentrated Sample Diluent (×4) 40 mL 1 vial

G Concentrated Protease Diluent (×10) 32 mL 1 vial

H Protease Powder 1.2 g 1 vial

［Reagent Preparations］

Transfer required volume of the reagents to sterile tubes, beakers, bottles, or other appropriate

container prior to use.

1. Sample Diluent Concentrated Sample Diluent (F); make a 4-fold dilution in distilled water. Sample Diluent is used to prepare standard solution, blank solution, and to dilute the protease-treated sample solution.

2. Protease Diluent Concentrated Protease Diluent (G); make a 10-fold dilution in distilled water. Protease Diluent is used to prepare protease solution

3. Protease Solution (It should be prepared within 15min before use) Add 20mL of the Protease Diluent to one vial of Protease Powder (H), and stir for 10min. at room temperature to make Protease Solution.

* Keep on ice immediately after preparation. ** If this kit is used in separate times, make aliquot of the Protease Solution and stock at –20°C

4. Sulfated GAG Standard Solutions Dilute the Sulfated GAG Standard (1,000!g/mL) (B) with Sample Diluent to make 6 standards (80, 40, 20, 10, 5, 2.5 !g/mL). Use Sample Diluent as blank solution (0!g/mL).

An example of the dilutions

Concentration 80 40 20 10 5 2.5 (!g/mL)

Sulfated GAG Standard (1,000!g/mL) 80 500 500 500 500 500 (!L)

Sample Diluent 920 500 500 500 500 500 (!L)

5. The other kit components are ready to use.

［Pre-treatment Procedure Summary］

Samples Tissue Chondrocyte Culture Sup.

Step 1 Volume 1～100mg 1～8×105cell 180!L

Step 2 Protease Diluent 1,800!L 180!L －

Step 3 Protease Solution 200!L 20!L 20!L

Step 4 Digestion 55°C, 20hrs. 55°C, 2hrs. 55°C, 2hrs.

Step 5 Stopping Reaction Boil for 10min.

Each assay step above should be performed without interruption.

Store protease-treated samples at 4°C if the assay cannot be done immediately after the

Pre-treatment. For long-term storage of pre-treated samples, store at -20°C.

［Assay Procedure Summary］

Each assay step above should be performed without interruption.

［Pre-treatment Procedure］

Notice: Pre-treatment procedure differs by samples.

!" Tissue Samples (a)： 1. Collect 1～100mg of tissue samples.

2. Add 1,800!L of Protease Diluent and 200!L of Protease Solution and then mix for 10sec.

3. Incubate for 20hrs. at 55°C and then boil for 10min.

!" Cultured Chondrocyte (b)： 1. Collect 1～8×105

cells of cultured chondrocyte.

2. Add 180!L of Protease Diluent and 20!L of Protease Solution and then mix for 10sec.


!" Cell Culture Supernatant (c)： 1. Collect 160!L of Culture Supernatant.

2. Add 20!L of Protease Diluent and 20!L of Protease Solution and then mix for 10sec.


［Assay Procedure］

Verify all reagents are at room temperature before commencing assay.

It is recommended that all standards and samples be assayed in duplicate.

1. Prepare all reagents, Sulfated GAG Standard Solutions and samples.*

2. Prepare Microplates （A）. 3. Add 50!L of Sulfated GAG Standard Solutions (80, 40, 20, 10, 5, 2.5!g/mL), blanks and/or

protease-treated samples.

4. [Tissue Samples(a) ]

Add 50!L of Reaction BufferⅠ(D) into each well.**

[Cultured Chondrocyte (b)] and/or [Cell Culture Supernatant (c)]

Add 50!L of Reaction BufferⅡ(E) into each well.**

5. Add 150!L DMMB Dye Solution (C) into each well.**

6. Incubate for 5 min. at room temperature (15-25°C) in the dark by covering the whole strips using aluminum foil.

7. Measure absorbance at 530nm using a microplate reader immediately.

* Remove particulates by centrifugation, and dilute with Sample Diluent if needed.

** Important Notice Multidispense pipette is recommended for accurate assay.

［Calculation of Sulfated GAG concentrations］

1. Average duplicate values for each Sulfated GAG standard and sample.

2. To make the Sulfated GAG standard curve, plot the Sulfated GAG concentration on the x-axis and the absorbance on the y-axis for each standard Sulfated GAG solution using graph paper or appropriate software. We recommend to use [quadratic] curve for the plot.

3. Determine the Sulfated GAG concentration for each diluted sample using the generated Sulfated GAG standard curve.

4. To obtain the Sulfated GAG concentration in each sample, multiply the above value by the appropriate dilution factor.

As to (diluted) samples that exceeded 80!g/mL, repeat the assay with proper dilution in Sample

Diluent.

［Typical Standard Curve］

［Precautions and Warnings］

1. Do not use this kit beyond the expiration date. 2. Do not mix the reagents with different lot numbers. 3. Bring all reagents and samples to room temperature before use. 4. Aliquot necessary amounts of all reagents prior to assay, to avoid possible contamination. 5. Perform entire assay without interruption. 6. Perform a Sulfated GAG standard curve for each assay. 7. Use all reagents within a week of opening the kit. 8. DMMB Dye Solution contains organic solvent and Reaction Buffers contains a protein-denaturing

reagent. If contact with skin or eyes occurs, rinse thoroughly with water and seek medical attention immediately.

［Storage］

2 - 8°C

For research use only.

Not for use in diagnostic procedures.

DMMB Dye Solution; 150!L

Measure the absorbance at 530nm

Reaction BufferⅠ or Reaction BufferⅡ; 50!L

Calculation of Sulfated GAG Concentration

Standard Solution or Protease-treated Samples; 50!L

Development Reaction: Room Temp, 5min, In Dark

Step 1

Step 2

Step 3

Step 4

0.40.60.81.01.21.4

0.000 0.020 0.040 0.060 0.080 0.100

Sulfated GAG Concentration (mg/mL)

22

RELATED PRODUCTS

Hyaluronan Assay Kit

This kit is based on a competitive inhibition method using hyaluronic acid binding protein (HABP) specific to Hyaluronan (HA) and is developed for measurement of HA in body fluids, culture medium, and tissue extracts.


［Contents of the Kit］

Contents Amount Number

A Hyaluronan-coated Microplate 8 well strip 12 strips

B Hyaluronan Standard (10!g/mL) 1 mL 1 vial

C Concentrated HRP-Conjugated Streptavidin Solution 0.3 mL 1 vial

D HRP-Conjugated Streptavidin Diluent 15 mL 1 vial

E Biotinylated HABP (Lyophilized) - 3 vial

F Biotinylated HABP Diluent 15 mL 1 vial

G Substrate (OPD; 1mg / tablet) - 3 tablets

H Substrate Buffer 15 mL 1 vial

I Stop Solution (1N HCl) 15 mL 1 vial

J Concentrated Sample Diluent (2X) 50 mL 1 vial

K Concentrated Wash Solution (5X) 50 mL 2 vials

L Frame (for Microplate Strips) 1 frame

M Cover Film (for Microplate) 3 films

［Reagent Preparations］

Transfer required volume of the reagents to sterile tubes, beakers, bottles, or other

appropriate container prior to use.

This protocol is stated for the assay of 4 strips.

Note that the volume of the reagent C and the Concentrated Biotinylated-HABP Solution required for the assay is differ for each lot of the kit.

1. Sample Diluent Concentrated Sample Diluent (J); make a 2-fold distilled water. Sample Diluent is used to prepare samples, HA standard solution, and blank solution.

2. Wash Solution Concentrated Wash Solution (K); make a 5-fold distilled water. Wash Solution is used to wash the microplate wells.

3. Biotinylated-HABP Solution (It should be prepared within 15min before use)

1) Add 500!L of the Biotinylated-HABP Diluent (F) to one vial of Biotinylated-HABP

(Lyophilized) (E). The reagent will take 10min to dissolve. And then, mix gently to make Concentrated Biotinylated-HABP Solution.

2) Add 160!L of Concentrated Biotinylated-HABP Solution prepared in step 1) to 4mL of Biotinylated HABP Diluent (F). Mix gently to make Biotinylated-HABP Solution.

4. HRP-Conjugated Streptavidin Solution

Add 30!L of Concentrated HRP-Conjugated Streptavidin Solution (C) to 4mL of

HRP-Conjugated Streptavidin Diluent (D). Mix gently to make HRP-Conjugated

Streptavidin Solution.

5. Substrate Solution (It should be prepared within 10min before use, avoiding light) Add one tablet of Substrate (G) to 4mL of Substrate Buffer (H) the reagent will take 5min to dissolve. Mix gently to make Substrate Solution. Avoid light throughout the preparation.

6. HA Standard Solutions

Dilute the HA Standard (10!g/mL)(B) with Sample Diluent to make 6 standards (400, 200,

100, 50, 25, 12.5 ng/mL). Use Sample Diluent as blank solution (0 ng/mL).


Final Concentrations 1000 400 200 100 50 25 12.5 (ng/mL)

HA Standard (10!g/mL) 100 400 500 500 500 500 500 (!L)

Prepared Sample Diluent 900 600 500 500 500 500 500 (!L)

7. The other kit components are ready to use.

［Assay Procedure Summary］

［Assay Procedure］



1. Prepare all reagents, HA Standard Solutions and samples. * **

2. Set the necessary number of strips of HA-coated Microplate (A) in the Frame (L).

3. Wash each well 3 times with 300 !L of Wash Solution.

4. Add 50 !L of HA Standard Solutions ( 400, 200, 100, 50, 25, 12.5ng/mL), blanks and/or samples.

5. Immediately, add Biotinylated-HABP Solution into each well and then mix gently for 1 min. using microplate-shaker. Incubate for 60 min. at 37°C. (Primary Reaction) ***

6. After removing the reaction solution, wash each well 3 times with 300 !L of Wash Solution.

7. Add 100 !L of HRP-Conjugated Streptavidin Solution into each well. Incubate for 60 min. at 37°C. (Secondary Reaction) ***

8. After removing the reaction solution, wash each well 5 times with 300 !L of Wash Solution.

9. Add 100 !L of Substrate Solution into each well. Incubate for 30 min. at room temperature (15

- 25°C) in the dark by covering the whole strips using aluminum foil. (Development Reaction) ***

10. Add 100 !L of Stop Solution (I) into each well and then mix gently for several seconds.

11. Measure absorbance at 492 nm using a microplate reader (reference wavelength, 630-650 nm).

* Remove particulates by centrifugation, and dilute with Sample Diluent as needed. ** Dilution number recommended for testing bodily fluids is as follows.

Serum Sample

Rat Rabbit

Conditioned medium

Dilution 2-fold or above 2-fold or above 2-fold or above

*** Seal with Cover Film (M) for microplate during the reaction.

［Calculation of Hyaluronan concentrations］

1. Average duplicate values for each HA standard and sample.

2. To make the HA standard curve, plot the HA concentration on the x-axis and the absorbance on the y-axis for each standard HA solution using graph paper or appropriate software. We recommend to use [logit-log] curve for x:log - y:linear plot or [quadratic] curve for x:log - y:log plot.

3. Determine the HA concentration for each diluted sample using the generated HA standard curve.

4. To obtain the HA concentration in each sample, multiply the above value by the appropriate dilution factor.

* As to (diluted) samples that exceeded 400ng/mL, repeat the assay with propedilution in Sample Diluent.

［Typical Standard Curve］

［Precautions and Warnings］

1. Do not use this kit beyond the expiration date. 2. Do not mix the reagents with different lot numbers. 3. Bring all reagents and samples to room temperature before use. 4. Aliquot necessary amounts of all reagents prior to assay, to avoid possible contamination. 5. Perform entire assay without interruption. 6. Perform a HA standard curve for each assay. 7. Use all reagents within a week of opening the kit. 8. Stop Solution contains 1N HCl. If contact with skin or eyes occurs, rinse thoroughly with water

and seek medical attention immediately.

［Storage］

2 - 8°C

NOTE



Standard Solutions ; 50 !L or Samples; 50 !L +

Biotinylated HABP Solution; 50 !L

HRP-Conjugated Streptavidin Solution; 100 !L

Substrate Solution; 100 !L

Stop Solution; 100 !L

Measure the absorbance at 492nm / 630 - 650nm

Primary Reaction: 37°C, 60 min

Secondary Reaction: 37°C, 60 min

Development Reaction: Room Temp, 30 min, in the Dark

Calculation of HA Concentrations

0.00.51.01.52.0

1 10 100 1000

A492 /

630 (lin

ear)

HA Concentration (ng/mL) (log)

RELATED PRODUCTS

23

RELATED PRODUCTSHeparan Sulfate ELISA Kit

This sandwich-type ELISA (Enzyme-Linked Immunosorbent Assay) kit is for measurement of Heparan

Sulfate (HS) using two specific monoclonal antibodies. This kit is developed for measurement of HS in

urine, culture media and other biological solutions.


Materials supplied with the Kit!!

Materials Amount Number

A Antibody-coated Microstrips 8 well strip 12 strips

B Heparan Sulfate Standard *1 (from human urine at 8µg/mL) 0.5 mL 1 vial

C HRP-Conjugated Streptavidin *2 12 mL 1 vial

D Biotinylated Antibody (Lyophilized)*3 - 1 vial

E Substrate (TMB) 12 mL 1 vial

F Stopping Solution 12 mL 1 vial

G Reaction Buffer Concentrate (5X) 40 mL 1 vial

H Washing Solution Concentrate (5X) 50 mL 2 vials

I Frame (for Microplate Strips) - 1 frame

J Cover Film (for Microplate) - 3 films

*1 HBs antigen test, HIV and HCV antibody test negative.

*2 Contains thimerosal at 0.0001%.

*3 Refer to "Reagent Preparations! for reconstitution volume.

Reagent Preparations!

Transfer required volume of the reagents to sterile tubes, beakers, bottles, or other

appropriate container prior to use.

All the reagents including prepared reagents for the assay should be used at room

temperature (RT: 15-25 °C).

1. Reaction Buffer Reaction Buffer Concentrate (G); Make a 5-fold dilution in distilled water.

Reaction Buffer is used to prepare samples, HS standards, and blank HS Solution.

2. Washing Solution Washing Solution Concentrate (H); Make a 5-fold dilution in distilled water.

Washing Solution is used to wash wells of microstrips, and is also used for

Biotinylated-Antibody preparation.

3. Biotinylated-Antibody Solution Dissolve completely Biotinylated-Antibody (Lyophilized) (D) with 12mL of the Washing

Solution.

4. HS Standard Solutions "STD range; 8-0.25µg/mL and blank (0µg/mL) !

Dilute the HS Standard (8µg/mL) (B) with the Reaction Buffer to make 5 standards (4, 2, 1,

0.5, 0.25µg/mL) . Use the Reaction Buffer as blank HS Solution (0µg/mL).


Final Concentrations ! ! 4 ! 2 ! 1 0.5 0.25 (µg/mL)

HS Standard (8 µg/mL)! 200! ! 200! 200 200 200 (µL)

Reaction Buffer ! ! ! ! 200! ! 200 200 200 200 (µL)

5. Other kit components are ready to use.

Summary of Assay Procedure!

"Assay Procedure!



1. According to the "Reagent Preparations], prepare diluted Reaction Buffer, Washing Solution,

samples*1 and Biotinylated-Antibody (reconstituted).

2. Set Antibody-coated Microstrips (A) as required in the Frame (I).

3. Wash wells of microstrips 5 times with 300µL of the Washing Solution.

4. Add 100µL of Reaction Buffer in each well. And then 20µL of HS Standard Solutions (8, 4,

2, 1, 0.5, 0.25µg/mL), blanks (0µg/mL), or samples and then mix gently*2. Incubate them for

18-24hrs. at 2-8 °C *3. (Primary Reaction)

5. Remove the contents of strips and discard *4, wash them 5 times with 300µL of the Washing

Solution.

6. Add 100µL of HRP-Conjugated Streptavidin (C) and 100µL of the Biotinylated-Antibody

(reconstituted) into each well and then mix gently*2. Incubate them for 60 min. at RT*

3.

(Secondary Reaction)

7. Remove the contents of strips and discard*4, wash the strips 5 times with 300µL of the Washing

Solution.

8. Add 100µL of Substrate (E) into each well and then mix gently*2. Incubate them for 30min. at

RT*3. (Color Development)

9. Add 100µL of Stopping Solution (F) into each well and then mix gently*2.

10. Measure the absorbance at 450nm in microplate reader (reference wavelength, 630nm).

*1 Samples with suspensions should be centrifuged to remove particles and then use the

supernatant. Use the Reaction Buffer for sample dilution.

< Examples of sample dilution >

Urine Sample

Rat Human Culture medium

Dilution # 1 / 2 # 1 # 1

*Serum requires pretreatment. Please contact us for detailed protocol.

*2 Gently shake the microstrips in the Frame (I) on a plate shaker or equivalent for a several

seconds to assure proper mixing (DO NOT splash).

*3 Seal the microstrips with Cover Film (J) during incubation.

*4 Discard the well contents to an appropriate container and pat the microstrips in the Frame

(I) on paper towels to remove the remainder.

"Calculation of Heparan Sulfate concentrations!

1. Average duplicate values for each HS standard and sample.

2. To make the HS standard curve (quadratic curve), plot the absorbance (blank subtracted) on the

x-axis (log) and the HS concentration on the y-axis (log) using graph paper or appropriate

software.

3. Determine the HS concentration for each sample using the generated HS standard curve.

4. To obtain the HS concentration in each sample, multiply the above value by the appropriate

dilution factor.

* As to (diluted) samples that exceeded 8µg/mL, repeat the assay with proper dilution in

Reaction Buffer.

"Typical Standard Curve!

"Precautions and Warnings!

1. Confirm expiry date before use.

2. Lot numbers of reagents used must be matched. Do not mix kit components from different

manufactured lots.

3. Verify all reagents are at room temperature before commencing assay.

4. Transfer and pipette all reagents aseptically. Caution must be taken when reconstituting kit

components so as not to mix the reagents.

5. For accurate results, the protocol must be completed within 2 consecutive days without

interruption.

6. Inappropriate incubation time and temperature can give a slight impact on the result accuracy.

Standard and samples should be run in the same plate.

7. Use all the reagents in the kit within a week once opened.

8. The HS component standard utilized in this kit is HBs antigen test, HIV, and HCV antibody test

negative. Use proper laboratory techniques when handling these reagents and dispose of

properly.

9. The Stopping Solution contains acidic material, use caution when handling (eye protection or

goggles if local guidelines required). If incidental contact does occur with skin or eyes, rinse

thoroughly with water and seek medical attention immediately.

"Storage!

2 - 8°C (in dark), DO NOT FREEZE

NOTE



!

Reaction Buffer; 100 µL +

Standard Solutions or Samples; 20 µL

HRP-Conjugated Streptavidin; 100 µL

Biotinylated Antibody Solution; 100 µL

Substrate; 100 µL

Stopping Solution; 100 µL

Measure the absorbance at 450nm / 630 nm

Primary Reaction: 2-8°C, 18-24hrs.

Secondary Reaction: RT, 60 min.

Color Development: RT, 30 min.

Calculation of HS Concentrations

HS Concentration (µg/mL)(log)

A450 /

630 (-B

lank)

(lo

g)

24

RELATED PRODUCTSHigh Sensitive Keratan Sulfate ELISAKit

This sandwich-type ELISA(Enzyme-Linked Immunosorbent Assay) kit is for measurement ofKeratan Sulfate(KS) using twoKS specificmonoclonal antibodies. This kit is developed for high sensitivemeasurement of KSin various animal samples, including serum and joint fluid which were undetectable with the previousmeasurement.


!Components of theKit"

Components Amount Number

A Antibody-coatedMicroplate 8well strip 12 strips

B Keratan SulfateStandard (fromshark cartilage at 8ng/mL) 0.4mL 1 vial

C ConcentratedHRP-ConjugatedAntibody(dryform) - 1 vial

D SubstrateSolution (TMB) 12mL 1vial

E Stop Solution (1mol/LHCl) 12mL 1 vial

F HRP-ConjugatedAntibodyDiluent 0.3mL 1vial

G ConcentratedReactionBuffer (5X) 40mL 1vial

H ConcentratedWashSolution (5X) 50mL 2vials

I Frame (forMicroplateStrips) - 1 frame

J Cover Film(forMicroplate) - 3 films

!Reagent Preparations"

Aliquot requiredvolume of reagents andpreparebeforeusing.Preparedreagentsmustbeused immediatelyandavoid storing.

1. ReactionBufferConcentrated Reaction Buffer (G);Make a 5-fold dilution in distilled water and prepare ReactionBuffer. This Reaction Buffer is used for dilution of KS standards and samples, and is also used as aBlank Solution.

2. HRP-ConjugatedAntibodySolution1) Add 200!L of HRP-Conjugated Antibody Diluent (F) to the vial of ConcentratedHRP-ConjugatedAntibody (C) andmixwell. (HRP-ConjugatedAntibodyConcentrateSolution)

2) Dilute HRP-Conjugated Antibody Concentrate Solution with to make a 101-fold dilution inReactionBuffer andmixwell (dilute at the date of use).(Ex.:ReactionBuffer; 6mL+HRP-ConjugatedAntibodyConcentrate Solution; 60!L)

3. WashingSolutionConcentratedWashSolution (H);Make a 5-fold dilution in distilledwater. ThisWashingSolution isused for washingmicroplates.

4. KSStandardSolutionsDilute theKS Standard (8ng/mL) (B) with the Reaction Buffer to make 5 standards (4, 2, 1, 0.5,0.25ng/mL).Use theReactionBuffer as blankKSSolution (0ng/mL).

Anexampleof thedilutions

FinalConcentrations 4 2 1 0.5 0.25 (ng/mL)

KSStandard (8ng/mL) 200 200 200 200 200 (!L)ReactionBuffer 200 200 200 200 200 (!L)

5. Other kit components are readytouse.

!Summary ofAssay Procedure"

Performentire assaywithout interruption.

!Assay Procedure"

Verify all reagents and samples are at room temperature (15-25 °C) before commencing the

assay.It is recommended that all standardsandsamples be assayed induplicate.

1. According to the !Reagent Preparations], prepare every required reagents, STD solutions andsamples*1,2.

2. SetAntibody-coatedMicroplate (A) as required in theFrame (I).

3. Add 100!LofReactionBuffer to eachwell.

4. And then add 20!L of KS Standard Solutions (4, 2, 1, 0.5, 0.25ng/mL), blanks (0ng/mL), orsamples to each well. Then Gently shake the microplate on a plate shaker or equivalent to assureproper mixing for approximately 30sec (DO NOT splash). Incubate them for 60min at roomtemperature. (PrimaryReaction)*3

5. Discard the well contents and wash the wells of microplate 5 times with 300!L of the WashingSolution

*4.

6. Add 100!L ofHRP-ConjugatedAntibodySolution to each well. Incubate them for 60min at roomtemperature. (SecondaryReaction)*3

7. Discard the well contents and wash the wells of microplate 5 times with 300!L of the WashingSolution

*4.

8. Add 100!L of Substrate Solution (D) to each well. Incubate them for 30min at room temperature(protect from light). (Color Development)*3

9. Add 100!LofStopSolution (E) to eachwell and thenmix gentlyfor a several seconds.

10. Gently shake the microplate on a plate shaker or equivalent to assure propermixing for approximately30sec (DO NOT splash). Then measure the absorbance at 450nm in microplate reader (referencewavelength, 630nm)within 30minof adding stopping solution.

*1 Remove particulates by centrifugation, and dilute the obtained supernatant with ReactionBuffer as needed.

*2Examples of sampledilution

SerumSample

Mouse Rat Guinea pig Rabbit Dog Human

Dilution x5 x50 x200 x50 x500 x1000

*3 Seal themicroplatewith PlasticFilm(J) during the incubation to avoid dryingout.

*4 For wash step, discard the well contents to an appropriate receptacle and rap the invertedmicroplate on absorbent paper towels to remove the remainder. Multi-channel manifolddispenser or equivalent is recommended to addWashingSolution intowells.

!Calculation ofKeratanSulfate concentrations"

1. Average duplicate values for eachKS standardand sample.

2. Tomake the KS standard curve (quadratic curve), plot the absorbance (blank subtracted) on the x-axis(log) and theKSconcentration on the y-axis (log) usinggraph paper or appropriate software.

3. Determine theKS concentration for each sample using the generatedKSstandard curve.

4. To obtain the KS concentration in each sample, multiply the above value by the appropriate dilutionfactor.

* As to (diluted) samples that exceeded 4ng/mL, repeat the assay with proper dilution in ReactionBuffer.

!Typical StandardCurve"

!Precautions andWarnings"

1. Donot use this kit beyond the expiration date.2. Donotmix the reagentswithdifferent lot numbers.3. Verifyall reagents are at room temperaturebefore commencing the assay.

4. Aliquot requiredvolume of reagents andprepare before using, and take care to avoid contamination.5. Performentire assaywithout interruption.6. PerformaKS standard curve for each assay.7. Use all reagentswithin aweekafter opening the kit.8. As theStoppingSolution contains acidicmaterial, usewith cautionwhenhandling (eye protection or

goggles if local guidelines required). If incidental contact does occurwith skin or eyes, rinse thoroughlywithwater and seekmedical attention immediately.

!Storage"

2 - 8°C (indark),DONOTFREEZE

NOTE

For researchuse only.

Not for use indiagnostic procedures.

ApplieBlock (CodeNo.: 200150)andQuick labeler Pro-Po/NH2(Code.No.: 200766) isused for thiskit.

KSConcentration (ng/mL)(log)

A450/630(-Blank)(log)

PrimaryReaction:RT, 60min

SecondaryReaction:RT, 60min

Color Development: RT, 30min

Calculation of KSConcentrations

ReactionBuffer; 100!L+

StandardSolution or Samples; 20!L

Step 1

HRP-ConjugatedAntibodySolution; 100!LStep 2

SubstrateSolution; 100!LStep 3

StopSolution; 100!LStep 4

Measure the absorbance at

450nm / 630nmStep 5

25

Standards and Substrates

Systematic Analysis of Glycosaminoglycan (GAG) Structure

Glycosaminoglycans

400640-1A Chondroitin, Na Salt Shark Cartilage 20 mg

400658-1A Chondroitin Sulfate A, Na Salt, Super Special Grade Sturgeon Notochord 2 mg

400660 Chondroitin sulfate B, Na Salt Pig Skin 20mg

400675-1A Chondroitin Sulfate C, Na Salt, Special Grade Shark Cartilage 2 g

400670-1A Chondroitin Sulfate C, Na Salt, Super Special Grade Shark Cartilage 20 mg

400676-1A Chondroitin Sulfate D, Na Salt, Super Special Grade Shark Cartilage 20 mg

400678-1A Chondroitin Sulfate E, Na Salt, Super Special Grade Squid Cartilage 2 mg

31254.01 Over Sulfated Chondroitin Sulfate Highly purified from 2 mg

31254.02 (OSCS) Standard Contaminated heparin sodium

10 mg

AMS.GAG-DS01 Dermatan Sulfate Pig Mucosa 2mg

31255.01 Dermatan Sulfate Standard Highly purified from 25 mg

31255.02 Impure heparin 100 mg

31255.03 sodium 250 mg

AMS.GM04-1 Sea Squirt Dermatan Sulfate Ascidiella aspersa 1mg

400700 Heparan Sulfate, Na Salt Bovine Kidney 2mg

AMS.GAG-HS01 Heparan Sulfate Pig Mucosa 2mg

AMS.GM01-1 Scallop Heparan Sulfate Pecten maximus 1mg

AMS.GM02-1 Whelk Heparan Sulfate Neptunea antiqua 1mg

400760 Keratan Sulfate, Na Salt Bovine Cornea 1mg

400720 Hyaluronic Acid, Na Salt Pig Skin 5mg

400763 Biotinylated Hyaluronic Acid Binding Protein Bovine Nasal Cartilage 50ug

See our technical bulletin “AMSBIO GAG Arrays” for suggestions of arrays that may be of particular value for investigation of protein-GAG interactions using AMSBIO’s Heparin/GAG Binding Plates.

Recommendations available for a) Glycosaminoglycan Array b) Heparin Oligosaccharide Array c) Heparin/HS Compatibility Array d) Sulfated K5 Polysaccharide Array Email [email protected] to find out more, or download a copy from www.proteoglycan.info

RELATED PRODUCTS

26

K5 Polysaccharides (semi-recombinant synthetic heparins and heparans)

Chemical analogues of heparin and heparan sulfate: Sulfated and spimerised derivitives.

K5 polysaccharide, produced in E. coli K5, has a repeat unit structure identical to the non-sulfated regions of heparan sulfate (HS); we provide this polymer in its native form and as low (L) and high (H) sulfated derivatives. Sulphation and epimerisation are carried out by chemical and enzymatic methods.

A selection of low molecular weight sulfated K5 materials is also available.

These modified K5 polysaccharides produce some of the characteristic structural features of heparin and heparan sulfate including the stepwise modifications that occur during polymer biosynthesis. However they also contain structural motifs that are not seen in HS and heparin. For example the K5/OS (H): (K5004) contains highly O-sulfated GlcA – GlcNAc repeat units and the E-K5/OS (H): (E-K5010) contains both O-sulfated IdoA – GlcNAc and GlcA – GlcNAc units. The most heparin-like material is the E-K5/NS, OS (H) (E-K5012) but this product has a lower iduronic content (approx. 50% of uronic acids are IdoA) and a higher O-sulphate content than heparin. See data sheet download for more information.

Sulfated K5 Polysaccharides

AMS.K5001 K5 Polysaccharide 1mg

AMS.K5002 K5/NS : N-Sulfated SO3-/COO- : 1.05 1mg

AMS.K5003 K5/OS(L) : O-Sulfated ,low SO3-/COO- : 1.55 1mg

AMS.K5004 K5/OS(H) : O-Sulfated, high SO3-/COO- : 2.63 1mg

AMS.K5005 K5/NS, OS(L) : N, O-Sulfated, low SO3-/COO- : 2.05 1mg

AMS.K5006 K5/NS, OS(H) : N, O-Sulfated, high SO3-/COO- : 3.78 1mg Epimerised (E) K5 derivatives

AMS.E-K008 E-K5/NS : N-Sulfated SO3-/COO- : 1.00 1mg

AMS.E-K009 E-K5/OS(L) : O-Sulfated, low SO3-/COO- : 1.16 1mg

AMS.E-K010 E-K5/OS(H) : O-Sulfated, high SO3-/COO- : 3.44 1mg

AMS.E-K011 E-K5/NS, OS(L) : N, O-Sulfated, low SO3-/COO- : 1.95 1mg

AMS.E-K012 E-K5/NS, OS(H) : N, O-Sulfated, high SO3-/COO- : 4.03 1mg

Heparin

24590.01 Heparin Sodium, research grade 500 mg

24590.02 Heparin Sodium, research grade 2.5 g

24590.03 Heparin Sodium, research grade 10 g

AMS.HEP001-100 Heparin - high grade 10mg

AMS.LCaHEP002-100 Low-In-Calcium Heparin 10mg

AMS.LMW Heparin Low Molecular weight heparin 10mg

AMS.HO22 Heparin Saccharide Mw. 7400 2mg



AMS.HO30 Heparin Polymer (>9000) 2mg

AMS.GM03-1 Crustacean Heparin (Nephrops norvegicus) 1mg

Selectively DeSulfated Heparins

These heparin products have been made from high quality heparin modified by standard chemical methods to selectively remove sulfate groups from C2 of Iduronate, (De2S Hep), C6 of glucosamine (De6SHep) or the N-sulfate of Glucosamine (DeNS Hep). The DeNS heparin contains the free amino group (NH

+3); in DeNS/Ac Hep the free amino group has been

modified by acetylation. This range of desulfated heparins is complemented by our series of sulfated K5 polysaccharides in which the internal uronate is glucuronic acid (GlcUA) in the native unmodified structure.

AMS.DSH001-2 2-O-Desulfated Heparin 2mg

AMS.DSH002-6 6-O-Desulfated Heparin 2mg

AMS.DSH003-N N-Desulfated Heparin 2mg

AMS.DSH004-NAc N-Desulfated reN-Aetylated Heparin 2mg

AMS.DSH005-pN Partially N-Desulfated Heparin 2mg

RELATED PRODUCTS

27

Oligosaccharide Standards

Dermatan Sulfate Oligosaccharides

Prepared from high quality Dermatan Sulfate (porcine origin) by partial GAG-endolyase scission and isolated by high resolution gel filtration.

General formula: !UA-GallNAc,4S – (IdoA – GalNAc,4S)n-IdoA-GalNAc,4S where n is number of disaccharide units n = 0 in a tetrasaccharide (dp4) n =1 in a hexasaccharide (dp6) n = 2 in an octasaccharide (dp8) etc…..

Uronic acid (!UA) at the non-reducing ends of the oligosaccharides has a C4-C5 double bond as a result of endolytic scission.

The main disaccharide in the original dermatan sulfate was IdoA – GalNAc,4S (88%) with minor quantities of 6-sulfated and 2,4 disulfated units (5% and 7% respectively) also present.

AMS.DSO04 Dermatan Sulfate dp4 1mg









Heparin Oligosaccharides

Prepared from high grade porcine heparin using bacterial Heparinase and isolated by high resolution gel filtration.

General formula:

!UA,2S-GlcNS,6S – (IdoA,2S – GlcNS,6S)n- IdoA,2S-GlcNS,6S

where 'n' is the number of disaccharide units n = 0 in the dp4 (HO04) tetrasaccharide n = 1 in the dp6 (HO06) hexasaccharide n = 2 in the dp8 (HO08) octasaccharide.........etc.

Uronic acid (!UA) at the non-reducing end of the oligosaccharides has a C4-C5 double bond as a result of the endolytic action of bacterial heparinase.

Although the main disaccharide unit in these products is IdoA,2S – GlcNS,6S (approx 75%) saccharides in each size class show some variation in sulfation.

AMS.HO04 Heparin dp4 2mg









RELATED PRODUCTS

28

Hyaluronic Acid Oligosaccharides

Hyaluronic Acid (HA) is a glycosaminoglycan composed of an alternating sequence of "1,3 glucuronic acid (GlcA) and "1,4 N-acetylglucosamine (GlcNAc). In its native state HA is normally present in the extracellular matrix as a high molecular weight, high viscosity polymer essential for maintenance tissue architecture, elasticity and hydration. However it also has other key functions including the regulation of cell behaviour through specific interactions with cell surface receptors and extracellular proteins. HA binding to individual proteins commonly involves relatively short sequences in the HA polymer and there is considerable evidence that HA fragments generated in vivo have distinctive properties from the intact polymer.

AMSBIO offers a range of oligosaccharides produced by controlled endolyase scission of purified, low endotoxin Hyaluronic Acid (Streptococcal species). The oligosaccharides are separated by high resolution gel filtration and purity assessed on an analytical Superdex S75 HPLC column (see Data Sheets for profiles). Size range of oligosaccharides dp2 to dp 20 dp is degree of monosaccharide polyimerisation: dp2 is a disaccharide, dp4 is a tetrasaccharide etc. General formula: !HexA"1,3 [GlcNAc"1,4 GlcA"1,3]n GlcNAc n = number of disaccharide units

!HexA is the C4-C5 unsaturated hexuronic acid at the non-reducing end of the oligosaccharides produced by endolyase scission of the HA polymer. The C4-C5 double bond absorbs strongly at 232nm and can be used for monitoring the oligosaccharides in various separation systems.

AMS.HA02 Hyaluronic Acid dp2 1mg









Disaccharide Standards

Disaccharide Sets and Mixtures

Unsaturated Chondro-Disaccharide Kit, Unsaturated Chondro-Disaccharide Kit (C-Kit) & Unsaturated Dermato/Hyaluro-Disaccharide Kit (D-Kit) contain unsaturated disaccharides with common structure: #4-unsaturated glucuronosyl-"-3-N-acetylhexosamine, with or without sulfated hydroxyl group(s). These unsaturated dissaccharides were separated & purified from the enzymatic digests of chondroitin sulfates A, C & D, dermatan polysulfate & hyaluronic acid by the action of eliminative cleavage of chondroitinase ABC & that of chondroitinase AC.

Unsaturated Heparan/Heparin-Disaccharide Kit (H Kit) and Unsaturated Heparan/Heparin-Disaccharide (H) Mixture contain unsaturated disaccharides with common structre : #4-unsaturated hexuronosyl-alpha-4-gluco-samine, with or without sulfated hydroxyl group(s) and/or sulfated amino the enzymatic digests of Heparin (Bovine intestine) , Chemically modified heparin1) and Heparan sulfate (e. g. heparan sulfate from bovine kidney ; cat. no. 400700 above ) by the action of eliminative cleavage of Heparitinases I and IV.

400570-1 Unsaturated Chondro-Disaccharide kit !Di-0S, !Di-4S, !Di-6S, 2 mgs/each.

1 kit

400571-1 Unsaturated Chondro-Disaccharide kit (C kit) !Di-0S, !Di-4S, !Di-6S, !Di-diSD, !Di-diSE, !Di-triS, 250 ug/each

1 kit

400572-1 Unsaturated Dermato/Hyaluro-Disaccharide kit !Di-HA, !Di-OS, !Di-4S, !Di-6S, !Di-UA2S, !Di-disB, ug/each.

1 kit

400575-1 Unsaturated Heparan Sulfate/Heparin-Disaccharide (H-kit) !DiHS-0S, !DiHS-NS, !DiHS-6S, !DiHS-diS1, !DiHS-diS2, !DiHS-triS. Each disaccharides(200nmole) supplied in separate tube.

1 kit

400576-1 Unsaturated Heparan Sulfate/Heparin-Disaccharide mixture (H) kit !DiHS-0S, !DiHS-NS, !DiHS-6S, !DiHS-diS1, !DiHS-diS2, !DiHS-triS. All disaccharides (100nmole supplied in single tube.

1 kit

RELATED PRODUCTS

29

Chondroitin/Dermatan Sulfate Disaccharides

Produced by bacterial chondroitinase digestion of Chondroitin and Dermatan sulfate.

Isolated by high resolution gel filtration and ion exchange chromatography.

Our product range includes the rare, but functionally important di- and

trisulfated Chondroitin/Dermatan sulfate disaccharides.

AMS.CD001 !UA – GalNAc 1mg

AMS.CD002 !UA – GalNAc,4S 1mg

AMS.CD003 !UA – GalNAc,6S 1mg

AMS.CD004 !UA – GalNAc,4S,6S (diE) 500ug

AMS.CD005 !UA,2S – GalNAc,4S (diB) 500ug

AMS.CD006 !UA,2S – GalNAc,6S (diD) 500ug

AMS.CD007 !UA,2S – GalNAc,4S,6S (triS) 500ug

AMS.CD008 !UA,2S – GalNAc 500ug

Heparin Disaccharides

Produced by the action of bacterial heparinase on high grade porcine heparin. Isolated by high resolution gel filtration and ion exchange chromotography

The uronate (!UA) contains a C4-C5 double bond due to the action of the heparinases used to depolymerise heparin

Our range includes N-unsubstituted disaccharides.

AMS.HD001 !UA,2S – GlcNS,6S 1mg

AMS.HD002 !UA,2S – GlcNS 1mg

AMS.HD003 !UA,2S – GlcNAc,6S 1mg

AMS.HD004 !UA – GlcNS,6S 1mg

AMS.HD005 !UA – GlcNS 1mg

AMS.HD006 !UA – GlcNAc 1mg

AMS.HD007 !UA,2S – GlcNAc 1mg

AMS.HD008 !UA – GlcNAc,6S 1mg

AMS.HD009 !UA-2S (R) GlcNCOEt-6S (internal standard Disaccharide) 1mg

Disaccharides with N-unsubstituted Amine

AMS.HD010 !UA,2S – GlcN 1mg

AMS.HD011 !UA,2S – GlcN,6S 1mg

AMS.HD012 !UA – GlcN,6S 1mg

AMS.HD013 Delta UA – GlcN 1mg

RELATED PRODUCTS

30

HA POLYMERS OF UNIQUELY DEFINED SIZES

Questions? We have answers!

Q: What is the

difference between

Select -HA™ and other

commercial HAs ?

A: The unique property of

Select -HA ™ is that it has very

narrow size distribution

while all other commercial

HA polymers are mixtures of

HA with a much broader size

range. Please see the

pictures (agarose gels

stained with Stains -All) for a

better understanding.

Select-HA™ of 285, 350, 425, 495 and 575 kDa(from left to right)

Other commercial HA of various sizes

Q: How are the sizes of Select -HA™ defined?

A: Due to the nature of the production process, there is lot -to -lot variation. If the

indicated molecular mass (determined by MALLS-SEC and reported on the Certificate of

Analysis) falls within 25-75 kDa , it is called Select -HA ™ 50. However, the Select -HA ™ 50 is

not a mixture of HA ranging from 25 kDa to 75 kDa. Remember, for any given lot, the

polydispersity is close to 1 ( i.e. close to monodispersity ).

Q: What is Select -HA™ ?

A: Select HA ™ is hyaluronic acid (HA) made through enzymatic synthesis where a very

high level of size control is achievable.

u Polydispersity as low as 1.02 and averages 1.1 (a value of 1 is for a ‘perfect’ polymer)

u Various sizes (25 kDa – 2500 kDa )u Choice of regular or low endotoxin , certified to be less than 0.1 EU/mg of HA

u Available biotinylated

u Custom orders possible

u Select -HA ™ 50 will be within the range of 25-75 kDa

u Select -HA ™ 150 will be within the range of 125 -175 kDa



u Select -HA ™ 1000 will be within the range of 800-1200 kDa


Select-HA™

31

RELATED PRODUCTS

SINGLE

-SIZE HA OLIGOMERS

Questions? We have answers!

Q: What is the chemical structure of nanoHA ™ ?

A: We currently offer nanoHA™ that consists of odd-numbered hyaluronate oligomers that

contain an N-Acetyl glucosamine unit at the reducing end.

Q: What is oligo -HA?

A: Oligo-HAs are purified single -size hyaluronic acid oligomers prepared by testicular

hyaluronidase digestion. Oligo -HA 4 is a monodisperse tetramer (4 monosaccharides :

Nonreducing -GlcUA -GlcNAc -GlcUA -GlcNAc -reducing end). Following that nomenclature,

oligo -HA 6 denotes the hyaluronic acid hexamer and oligo -HA 8 the octamer .

Q: What is nanoHA ™ ?

A: nano refers to our line of small oligomers where sugars are addedchemoenzymatically

to an oligo -HA precursor in a controlled step -wise fashion to produce small HA

oligosaccharides. Reminiscent of the oligo nomenclature, nanoHA5™ is five

monosaccharide units in length.

Q: What is the difference between nanoHA™ and Oligo -HA?

A: For a given size (e.g. oligo -HA 6 and nanoHA 6) both oligosaccharides are structurally

identical. The terms “oligo ” and “nano ” refer to the different procedures through which

each product is obtained. “oligo -HA ” is obtained via enzymatic digestion of hyaluronan

polymer with testicular hyaluronidase , while “nanoHA ” is obtained by stepwise

chemoenzymatic extension of oligoHA with UDP -GlcUA and UDP -GlcNAc , respectively.

Currently, we offer odd -numbered nanoHAs synthesized by a single sugar addition to

their precursor.

Q: What is the chemical structure of oligo -HA?

A: Oligo -HA consists of even -numbered hyaluronate oligomers that contain an N-Acetyl

glucosamine unit at the reducing end.

OOHO

OH

CO2H O

HOOH

OCO2H

OO

O

NHAc

HOOH

O

NHAc

HO

OH

OH

n

n = 1 nanoHA-5

n = 2 nanoHA-7

n = 3 nanoHA-9HO

O

NHAc

HO

OH

OHO

HOOH

CO2H O

HOOH

OCO2H

OO

O

NHAc

HOOH

O

NHAc

HO

OH

OH

n

n = 1 oligoHA-4

n = 2 oligoHA-6

n = 3 oligoHA-8

Oligo-HA & nanoHA

RELATED PRODUCTS

32

Hyalose Ladders

Select -HA™ HiLadder - The HiLadder contains five Select-HA™ molecular mass markers in the range of

~500 kDa to ~1500kDa. Recommended usage: size standard for gel electrophoresis.

Mega -HA™ Ladder - The Mega-HA™ Ladder is a mixture of streptavidin complexes containing one,

two, three or four end-labeled biotin-Select-HA™ molecules of very defined sizes for use as size standards in gel electrophoresis or other separation methods. This ladder covers a range from 2 MegaDalton to 8 MegaDalton. Recommended usage: size standard for gel electrophoresis.

NanoHA10-20™ Ladder – The NanoHA10-20™ Ladder is a preparation of different sizes of hyaluronic

acid oligosaccharides. The reducing end contains N-acetylglucosamine. This ladder contains oligomers of HA10, HA12, HA14, HA16, HA18, and HA20. Recommended usage: acrylamide gel stained with Alcian blue 8Gx or silver stain.

Select -HA™ LoLadder - The LoLadder contains five Select-HA™ molecular mass markers in the range of

~25 kDa to ~500 kDa. Recommended usage: size standard for gel electrophoresis.

495 kDa

572 kDa

966 kDa

1090 kDa

1510 kDa

Electrophoretic Separation of Select-HATM HiLadder Standards.Five µl of reconstituted HiLadder (Lot HL0503) was separated on a 1.0% (w/v) agarose gel by electrophoresis and stained with Stains-All.

495 kDa

310 kDa

214 kDa

110 kDa

27 kDa

Electrophoretic Separationof Select-HATM LoLadder StandardsFive µl of reconstituted LoLadder (Lot LL0401) was separated on a 1.0% (w/v) agarose gel by electrophoresis and stained with Stains-All.

6100 kDa

4570 kDa

3050 kDa

1520 kDa1510 kDa

ElectrophoreticSeparation of Mega-HATM

0.25 • g of Mega-HATM

Ladder (Lot ML200505, right) was separated on a 1% (w/v) agarose gel by electrophoresis and stained with Stains-All. Left, Select-HA™ HiLadder Lot: HL0503.

1

2

3

4

5

6

1 : HA20

3811.2 Da2: HA

18 3431.9 Da

3 : HA16

3052.6 Da4 : HA

142673.2 Da

5 : HA12

2293.9 Da6 : HA

101914.6 Da

SIZE STANDARDS FOR GLYCOBIOLOGY RESEARCH

RELATED PRODUCTS

33

Cat. No. Description Size

Select-HA™ Ladders

HYA-NALAD-20 � ��

HYA-LOLAD-20 ��

��

HYA-HILAD-20 ��

��

HYA-MGLAD-20 ��

��

Select-HA™

�� !"�#��

��'(�� !"�#��)�'�� *��

�� !"�#��

��'(�� !"�#��)�'�� *��

�� !"�#��

��'(�� !"�#��)�'�� *��

�� !"�#��

��'(�� !"�#��)�'�� *��

�� !"�#��

��'(�� !"�#��)�'�� *��

�� !"�#��

Biotinylated Select-HA™

��,�� !"�#�,� �-$��

��,�� !"�#�,� �-$��

��,�� !"�#�,� �-$��

��,�� !"�#�,� �-$��

Oligo-HA™

��'(�� )�'�� *�� $�

��%'(�� %��)�'�� *�� $�

��&'(�� &��)�'�� *�� $�

��'(�� )�'�� *�� $�

Nano-HA™

��'(�� !"�#��)�'�� *�� $�

��.'(�� !"�#�.��)�'�� *�� $�

��'(�� !"�#��)�'�� *�� $�

Select-HA™ Hyaluronic Acid polymers of uniquely

defined sizes

Mol. W

Oligos HA10 to HA20.

~25 kDa to ~500 kDa.

~500 kDa to ~1500kDa.

2 to 8 MegaDalton ��

��$�

��$�

��$�

25-75 kDa

25-75 kDa

125-175 kDa

125-175 kDa ��$�

��$�

��$�200-300 kDa

200-300 kDa

��$�400-600 kDa

��$�400-600 kDa

��$�800-1200 kDa

��$�800-1200 kDa

��$�2250-2750 kDa

~776.7 Da

~1155.97 Da

~1535.3 Da

~1919.1 Da

~979.84 Da

~1359.16 Da

~1738.47 Da

RELATED PRODUCTS

34

Heparin/GAG Binding Plates

Cat. No. AMS.H-G Plates Description Heparin/GAG Binding Plates ((96 well) Pack size: 5 plates per box

Surface immobilisation of native glycosaminoglycans (GAGS) in a 96-well format. ! No modification of GAG necessary.

! Protein binding properties retained.

! Versatile and simple to use.

! Amenable to high throughput screening.

! Binds GAGs at room temperature using physiological buffers.

! Binds a wide range of GAGs; from fully sulfated heparin to non-sulfated hyaluronan from high molecular weight polysaccharides down to decasaccharides.

Use of Heparin/GAG Binding Plate for detection of IL8 bound to Heparin

Product Specification

Microplate Type: Clear 96-well configuration complies with ANSI/SBS standards.

5 individually packaged plates per pack

Stable for at least 6 months when stored dry at 4 - 30 C

Quality Control: Uniform binding distribution of heparin to each well via labelled bioassay.

See our technical bulletin “AMSBIO GAG Arrays” for suggestions of arrays that may be of particular value for investigation of protein-GAG interactions using AMSBIO’s Heparin/GAG Binding Plates (pXX)

Recommendations available for

a) Glycosaminoglycan Array b) Heparin Oligosaccharide Array c) Heparin/HS Compatibility Array d) Sulfated K5 Polysaccharide Array

Email [email protected] to find out more, or download a copy from www.proteoglycan.info

RELATED PRODUCTS

35

Enzyme Activity Assays AMSBIO supply Razie assay kits for quantitative detection of Heparanase and Hyaluronidase in cell culture supernatants, human plasma, biological fluids and tissue samples.

Kit features

! Suitable for inhibitor screening

! Non-radioactive

! Fast and easy to use

! Sensitive and specific

! Uses a universal 96-well plate format ideal for inhibitor studies

Heparanase Assay Kit

Ra001- BE-K Heparanase Kit with Bacterial Enzyme as control 96 rxns

Ra001-02-K Heparanase Kit without enzyme 96 rxns

A handicap in Heparan Sulfate research has been a lack of a sensitive and more importantly specific test for human eparanase activity. Furthermore unavailability of a purified enzyme or instability of the cloned enzyme limits assay design. To date the available tests have the above shortcomings and are time consuming, not applicable for inhibitor creening or lack an appropriate positive control.

Hyaluronidase Assay Kit

Ra003-01-HAK Hyaluronidase Kit 96 rxns

For screening of Hyaluronidase inhibitors, and quantification of Hyaluronidase activity

PRINCIPLES OF THE TEST (HEPARANASE ASSAY)

GAG-coated 96-well plates available separately

FB001-01-P- HS HS-plate (Component of Ra001-BE-K & Ra001-02-K above)

FB001-02-HiS-P High sensitivity HS Plate

Ra1001-03-HSP HS Plate (Higher concentration of bounded Biotynylated HS)

Ra1001-04-NBP HS Plate (Higher concentration of bounded HS Non biotinylated)

Ra1002-01-HEP Heparin Plate (heparin bounded plate, not biotynylated)

Ra1003-01-HAP Biotinylated hyaluronic acid (hyaluronan) plate

Some Hs is left in the well post heparanase treatment

Wash

Heparanase treatment

A B C

Hs fragments washed away

Native well with biotinylated HS covalently bound

Streptavidin-HRP added

Wash

HRP substrate is added

D Streptavidin-HRP binds to the Biotion

Colour is generated after addition of HRP substrate

Decrease in the OD of well D compared to A is directly proportional to heparanase activity.

RELATED PRODUCTS

36

GAG Degrading Enzymes

Purified GAG Degrading Enzymes

100350-1A Chondro-4-Sulfatase (Proteus vulgaris) 1.6 units

100355-1A Chondro-6-Sulfatase (Proteus vulgaris) 2.5 units

100330-1A Chondroitinase ABC (Proteus vulgaris) † 10 units

100332-1A Chondroitinase ABC Protease Free (Proteus vulgaris) † 2 units

100334-1A Chondroitinase AC I Flavo (Flavobacterium heparinum) 1 unit

100334-2 Chondroitinase AC I Flavo (Flavobacterium heparinum) 50 units

100335-1A Chondroitinase AC II Arthro (Arthrobacter aurescens) 5 units

100335-2 Chondroitinase AC II Arthro (Arthrobacter aurescens) 2 units

100337-1A Chondroitinase B (Flavobacterium heparinum) 0.1 unit

100700-3 Heparinase (Flavobacterium heparinum) 0.1 unit

100703-3 Heparitinase (Flavobacterium heparinum) ‡ 0.1 unit

100704-1A Heparitinase I (Flavobacterium heparinum) 0.1 unit

100705-1 Heparitinase II (Flavobacterium heparinum) 0.1 unit

25118.01 Hyaluronidase (ovine testes) 50mg

25118.02 Hyaluronidase (ovine testes) 500mg

100740-1 Hyaluronidase (Streptomyces hyalurolyticus) 4 ampules

100741-1A Hyaluronidase SD (Streptococcus dysgalactiae) 0.5 units

100810-1 Keratanase (Pseudomonas sp.) 10 units

100812-1 Keratanase II (Bacillus sp.) 0.1 unit

† Chondroitinase ABC: 100332 is of higher purity than 100330 (and protease free). Difference in terms of applications is as

follows:

! 100330 can be used for selective removal of CS or DS side chains from proteoglycans, yielding a protein-enriched core molecule, which will display characteristic stubs, but will also have been subject to some damage from proteases.

! 100332 can be used to prepare or analyse proteoglycan core proteins at high level of purity (and undamaged by protease activity)

‡ 100703-3 (Heparitinase from F. heparinum, 0.1U) is a mix of 100704-1A (Heparitinase I) and 100705-1 (Heparitinase II).

Mixing ratio is 4(100704):1(100705).

Recombinant GAG Degrading Enzymes

AMS.50-013 Chondroitinase AC, from Recombinant Flavobacterium heparinum - Research Grade 0.5 IU

AMS.50-018 Chondroitinase B from Recombinant Flavobacterium heparinum - Research Grade 5 µg

AMS.50-010 Heparinase I from Recombinant Flavobacterium heparinum - Research Grade 0.5 IU

AMS.50-010-001 Heparinase I from Recombinant Flavobacterium heparinum - Research Grade 0.1 IU

AMS.50-011 Heparinase II, from Recombinant Flavobacterium heparinum - Research Grade 0.5 IU

AMS.50-011-001 Heparinase II, from Recombinant Flavobacterium heparinum - Research Grade 0.1 IU

AMS.50-012-001 Heparinase III from Recombinant Flavobacterium heparinum - Research Grade 0.1 IU

AMS.50-012 Heparinase III, from Recombinant Flavobacterium heparinum - Research Grade 0.5 IU

AMS.HL01 K5 Heparan Lyase (NEW) * 0.1 IU

* K5 Heparan Lyase (AMS.HL01) cleaves heparan sulfate in the non-sulfated regions of the polymer chain. This action may release activities that lie cryptic in the intact heparan sulphate chain. Can be used to remove heparan sulfate from proteoglycans and it is the only enzymatic method available for excising the entire sulfated regions from heparan sulfate chains. It differs from heparinase III (heparitinase I) in that it will not degrade the transition zones that have an intermediate level of sulfation. (Murphy et al. (2004), J. Biol. Chem. 279: 27239-27245).

An excellent substrate for this enzyme is K5 polysaccharide (AMS.K5001) which can be used to monitor the progress of the enzyme action by measuring absorbance at 232nm.

Alternative Names for GAG-degrading enzymes Cat. No. (Purified) Classification Alternative Classification Cat. No. (Recombinant) 100700-3 Heparinase is equivalent to Heparinase I AMS.50-010 100703-3 Heparitinase is equivalent to Heparinase II and III 100704-1 Heparitinase I is equivalent to Heparinase III AMS.50-012 100705-1 Heparitinase II is equivalent to Heparinase II AMS.50-011

RELATED PRODUCTS

37

GAG Degrading Enzymes / Substrates

Action of different GAG Degrading Enzymes

Heparitinase I & Chondroitinase ABC K5 Heparan Lyase

RELATED PRODUCTS

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