1Glycosaminoglycans Assay Kit
Catalog # 6022
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify GAGs

FORMAT:

96-well ELISA plate with removeable strips

ASSAY TYPE:

Colorimetric assay

ASSAY TIME:

5 minutes

STANDARD RANGE:

50 µg/ml to 3.1 µg/ml

NUMBER OF SAMPLES:

Samples NOT containing extra proteins: up to 42 (duplicate) samples/plate
Samples containing extra proteins: up to 21 (duplicate) samples/plate

SAMPLE TYPES:

Tissue homogenate

RECOMMENDED SAMPLE DILUTIONS:

VariesCHROMOGEN:

N/A (read at 525 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (1.7-7.6%)/Inter-Assay (5.2-8%)/Spiking Test (106-117%)

NOTES:

This assay uses a 5-point standard curve
Samples need to be solubilized before running assay© 2023 Chondrex, Inc. All Rights Reserved, 6022 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2

Glycosaminoglycans Assay Kit
Catalog # 6022
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Glycosaminoglycans (GAGs) are negatively charged polysaccharides located in most connective tissues and extracellular matrices (ECM),
as well as on the surfaces of many cell types. Consisting of repeating core disaccharide units, GAGs are categorized into four types:
heparan/heparan sulfate, chondroitin/dermatan sulfate, keratin sulfate, and non-sulfated hyaluronan. Sulfated GAGs in the ECM exist as
proteoglycans which typically consist of multiple glycosaminoglycan chains attached to a core protein (1). In a highly organized ECM, articularcartilage is composed of type II collagen, hyaluronan, link protein, and chondroitin sulfate-rich proteoglycans, which provide the osmotic
resistance necessary for cartilage to resist compressive loads (2).
In addition to type II collagen, GAGs are considered an autoantigen of rheumatoid arthritis (RA), as anti-GAGs antibodies associated with
ECM degradation exist in the serum of RA patients (3). Although the role of proteoglycans in RA is still unknown, research shows that
immunizing mice with proteoglycans consisting of GAGs can induce arthritis (4). Similarly, the loss of ECM is the first observable change inosteoarthritis (OA), therefore, determining GAGs levels may be a useful marker of disease progression. Furthermore, the successful use of
artificial cartilage for the treatment of OA necessitates the analysis of type II collagen and GAGs content to ensure the cartilage quality (5).
Chondrex, Inc. provides a sulfated GAGs Assay Kit (Cat # 6022) using cationic dye 1,9 dimethylmethylene blue (DMB) which binds to highly
charged sulfated GAGs, not including hyaluronan (6). This kit utilizes an improved DMB solution, minimizing interference with negatively
charged contaminants such as DNA and RNA and uses chondroitin sulfate as a standard for the analysis of ECM in cartilage. Moreover, toanalyze collagen coexisting with GAGs in cartilage, Chondrex, Inc. provides a variety of type and species-specific collagen detection kits
and total collagen detection kits. For more information, please visit www.chondrex.com or contact us at support@chondrex.com.

KIT COMPONENTS
Item

Quantity

AmountStorage

Chondroitin-Sulfate Standard (60221)
1,9 Dimethylmethlyene Blue (DMB) Dye Solution (60222)
PBS (60223)
ELISA Plate

1 vial
1 bottle
1 bottle
1 each

0.5 mg/ml, 0.5 ml
10 ml
50 ml

-20°C
-20°C
-20°C
-20°C

96-well (8-well strips x 12)

ASSAY OUTLINE
Add 100 µl of diluted standards and samples to wells

Add 100 µl of dye solution to wells (PBS for sample blanks)

Read at 525 nm WITHIN 5 MINUTES OF MIXING© 2023 Chondrex, Inc. All Rights Reserved, 6022 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

PLATE MAPPING
Assay Layout 1 - Samples NOT Containing Proteins

Assay Layout 2 - Samples Containing Proteins
123456789101112A

5050

S1S1

SB1

SB1

S9

S9

SB9

SB9S17

SB17S36B

2525

S2

S2

SB2

SB2

S10

S10

SB10 SB10

S17

SB17

S37

S37

C

12.5

12.5

S3

S3

SB3

SB3

S11

S11

SB11 SB11

S18

SB18

S30

S38

S38

D6.3

6.3

S4

S4

SB4

SB4S12S12

SB12 SB12

S18

SB18

S31

S31

S39

S39

E

3.1

3.1

S5

S5SB5SB5S13S13

SB13 SB13

S19SB19S24S32

S32

S40

S40FBBS6S6SB6SB6S14S14

SB14 SB14

S19SB19S25

S25

S33

S33

S41

S41GS21S21

S7S7SB7SB7S15

S15

SB15 SB15

S20

SB20

S26

S26

S34

S34

S42

S42H

SB21 SB21

S8S8

SB8

SB8

S16

S16

SB16 SB16

S20

SB20

Standards

Samples123456789101112A5050

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

B

2525

S4S4S12S12S20

S20

S28

S28

S36

C12.512.5

S5S5S13

S13

S21

S21

S29S29D6.36.3

S6

S6

S14

S14

S22

S22

S30

E3.1

3.1

S7

S7

S15

S15

S23

S23FBB

S8S8S16

S16

S24

GS1

S1S9S9S17S17H

S2

S2

S10

S10

S18

S18StandardsSamples

Sample Blanks

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placingthe bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: Samples need to be solubilized. Please inquire at support@chondrex.com for Chondrex, Inc.’s “Tips on GlycosaminoglycanSolubilization”
NOTE 7: Guanidine used for extracting GAGs from samples will interfere with this assay. Guanidine extracted samples should be diluted or
dialyzed against PBS to reduce the guanidine concentration to less than 0.25 M. In addition, some proteins have an absorbance at 525
nm; therefore, samples contaminated with unnecessary proteins must be diluted to a 5% or less protein solution and require sample blanks
to ensure accurate results. Please refer to assay layout 2 for samples containing proteins.

© 2023 Chondrex, Inc. All Rights Reserved, 6022 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: Take 50 µl of standard solution and mix with 450 µl of PBS (50 µg/ml). Then serially dilute the 50 µg/ml
standard stock solution with PBS. For example, mix 250 µl of the 50 µg/ml standard stock solution with an equal volume of PBS tomake a 25 µg/ml solution, and then repeat it three more times for 12.5, 6.3, and 3.1 µg/ml solutions. Chondrex, Inc. recommends
making fresh serial dilutions for each assay.
50 ml

500 mg/ml

2.

250 ml

250 ml

250 ml

250 ml123456450 ml

250 ml

250 ml

250 ml

250 ml

250 ml50 mg/ml

25 mg/ml

12.5 mg/ml

6.3 mg/ml

3.1 mg/ml

Blank

Prepare Sample dilutions: Sample dilutions will vary depending on the source or preparation protocol of samples. Two to threedifferent sample dilutions with PBS are recommended if the GAGs levels in the samples are unknown.
3.

Add Standards and Samples: Choose 3-1 or 3-2 depending on your samples.
3-1. Samples NOT containing extra proteins (use assay layout 1): Add 100 µl of standards, PBS (blank, B) into the purplewells, and samples into the orange wells in duplicate. For example, add 100 µl of sample 1 into the S1 wells, and then add 100 µlof sample 2 into the S2 wells. Proceed to Step 4-1.
3-2. Samples containing extra proteins (use assay layout 2): Add 100 µl of standards, PBS (blank, B) into the purple wells,and samples into the orange and gray wells (SB) in duplicate. For example, add 100 µl of sample 1 into the S1 and SB1 wells,and then add 100 µl of sample 2 into the S2 and SB2 wells. Proceed to Step 4-2.

4.

Add Dye Solution: Choose 4-1 or 4-2 depending on your samples.
4-1. Samples NOT containing extra proteins (use assay layout 1): Add 100 µl of Dye Solution into all wells.
4-2. Samples containing extra proteins (use assay layout 2): Add 100 µl of Dye Solution into the purple and orange wells andadd 100 µl of PBS into the gray wells (SB).

5.

Read Plate: Read the plate at 525 nm (or 530 nm) within 5 minutes after performing step 4.

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank (B), standards, test samples, and sample blanks (if used).

2.

Subtract the averaged blank OD values from the averaged OD values of the standards and test samples.
NOTE: If sample blanks are used, subtract averaged “sample blank” (SB) OD values from the averaged OD values of the corresponding
test samples in Step 1

3.

Plot the OD values of standards against the concentration of chondroitin-sulfate (µg/ml). Using a log/log plot will linearize the data.
Figure 1 shows a representative experiment where the standard range is 3.1 - 50 µg/ml.

4.

Chondroitin-sulfate concentration in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor
to obtain the chondroitin-sulfate concentration (µg/ml) in the original sample specimens. For additional assistance, please download asample calculation worksheet from www.chondrex.com.

© 2023 Chondrex, Inc. All Rights Reserved, 6022 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.comsupport@chondrex.com

5Figure 1 - A Typical Standard Curve for the Glycosaminoglycans Assay Kit
1.00

OD 525 nm

R² = 0.9936

0.100.01
110100Chondroitin-6-Sulfate (mg/ml)

ASSAY VALIDATION
Table 1 - Reproducibility Data for the Glycosaminoglycans Assay Kit
Test
6.3 µg/ml
12.5 µg/ml
25 µg/ml
Intra-Assay CV (%)
7.6
3.6
1.7
Inter-Assay CV (%)
7.3
5.2
8.0
Spike Test* (%)
116%
117%
106%
*Known amounts of chondroitin-sulfate were added to standards and then diluted with PBS for assaying GAGs.

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

J. Yoon, J. Halper, Tendon Proteoglycans: Biochemistry and Function. J Musculoskelet Neuronal Interact 5, 22-34 (2005).

2.

J. Buckwalter, H. Mankin, Articular Cartilage: Tissue Design and Chondrocyte-Matrix Interactions. Instr Course Lect 47, 477-86 (1998).

3.

J. Wang, M. Roehrl, Glycosaminoglycans Are a Potential Cause of Rheumatoid Arthritis. Proc Natl Acad Sci U S A 99, 14362-7 (2002).

4.

B. Farkas, F. Boldizsar, O. Tarjanyi, A. Laszlo, S. Lin, et al., BALB/c Mice Genetically Susceptible to Proteoglycan-Induced Arthritis andSpondylitis Show Colony-Dependent Differences in Disease Penetrance. Arthritis Res Ther 11, R21 (2009).

5.

Q. Wang, N. Hughes, S. Cartmell, N. Kuiper, The Composition of Hydrogels for Cartilage Tissue Engineering Can Influence
Glycosaminoglycan Profile. Eur Cell Mater 19, 86-95 (2010).

6.

I. Barbosa, S. Garcia, V. Barbier-Chassefière, J. Caruelle, I. Martelly, D. Papy-García, et al., Improved and Simple Micro Assay for
Sulfated Glycosaminoglycans Quantification in Biological Extracts and Its Use in Skin and Muscle Tissue Studies. Glycobiology 13,647-53 (2003).

© 2023 Chondrex, Inc. All Rights Reserved, 6022 2.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com