1Collagenase Assay Kits
Catalog # 3001 & 3002
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to assess collagenase activity
3001: Rapid Collagenase Assay Kit with Type I Collagen Substrate
3002: Rapid Collagenase Assay Kit with Type II Collagen Substrate

FORMAT:

96-well ELISA Plate with non-removeable strips

ASSAY TYPE:

Enzyme Assay/Fluorescence-based Assay

ASSAY TIME:

2.5 hours

STANDARD RANGE:

Depends on incubation time

NUMBER OF SAMPLES:

Up to 44 (duplicate) samples/plate

SAMPLE TYPES:

Culture Media and Tissue HomogenateRECOMMENDED SAMPLE DILUTIONS:

Depends on enzyme activity in samples

CHROMOGEN:

N/A (Read at Emission 520 nm/Excitation 490 nm)

STORAGE:-20°C for 12 months (Reference Standard is stored at -80°C)

VALIDATION DATA:N/ANOTES:

Uses FITC© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

Collagenase Assay Kits
Catalog # 3001 & 3002
For Research Use Only - Not Human or Therapeutic Use
INTRODUCTION
Collagenases are members of the matrix metalloproteinase (MMP) family and degrade collagen types I, II, and III. At least three distinct
forms of collagenase (MMP-1, MMP-8, and MMP-13) have been identified. Collagenases are produced by many types of cells such as
myeloid and fibrosarcoma cells. Increased collagenase levels have been found in physiological conditions, such as post-partum uterine
tissue or tadpole metamorphosis, and pathological conditions, such as inflammation and tumor metastasis. These collagenases have almostidentical substrate specificities. However, individual collagenases may have unique enzyme-substrate affinities, resulting in different
physiological and pathological roles in the turnover of collagen depending on the tissues and cell types. For example, MMP-13 digests type
II collagen ten times faster than type I collagen.
This kit is designed to assay mammalian collagenase activities in approximately two hours using FITC-labeled telopeptide-free soluble bovine
type I collagen (Catalog # 3001) or bovine type II collagen (Catalog # 3002) as a substrate (1). This kit only requires 1/10 of the assay timeof conventional assay methods that use a collagen gel substrate and it also has higher assay sensitivity than assays using radioisotopelabeled collagen (2).

KIT COMPONENTS
Item

Quantity

AmountStorage

Reference - Recombinant Human MMP-8 (30043)
Solution A - FITC-Collagen Dilution Buffer (30041)
Solution B - Sample Dilution and Reaction Buffer (30042)
FITC-Collagen - 2X FITC-Labeled Bovine Type I or Type II
Collagen (4001 or 4002)
Activator 1 - 20X APMA (Toxic - Handle with Care) (30049)
Activator 2 - Trypsin (30045)
Proteinase Inhibitor (30046)
Enhancer - Elastase (30047)
Stop Solution - o-Phenanthroline (300410)
Extraction Buffer (30048)

1 vial
1 bottle
1 bottle

25 µl, 100 units/ml
10 ml
50 ml

-80°C
4°C
4°C

1 bottle

10 ml, 1 mg/ml-20°C

1 vial1 vial1 vial1 vial1 vial2 bottles

1 ml
1 mg lyophilized
3 mg lyophilized
1 mg lyophilized
1 ml, 10 mM
25 ml-20°C/*
-20°C
-20°C
-20°C
-20°C/*-20°C/*

2 each

96-well-20°C/*

Black ELISA Plate*These reagents can also be stored at room temperature© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

PRINCIPLE OF THE RAPID COLLAGENASE ASSAY KIT (TYPE I COLLAGEN)
Mammalian collagenases cleave the alpha chain triple helixes of collagen, yielding 3/4 and 1/4 collagen fragments, TC A and TCB fragments.
The denaturation temperature of these fragments is 34-35°C and 28-29°C respectively, whereas the denaturation temperature of intactcollagen is 40°C. Therefore, these cleaved fragments selectively denature into single random coils at 35°C which can be extracted with
Extraction Buffer. To shorten the denaturation process, which normally takes 60 minutes, an enhancer is used to further digest collagenasedegraded products into small peptides (2).

This assay consists of four steps:
1.

Activate latent collagenase in samples with an Activator

2.

React the activated samples with FITC-labeled soluble
collagen for 10-120 minutes

3.

Denature and further digest the cleaved collagenfragments into small peptide fragments by Enhancer

4.

Extract the fragments with Extraction Buffer anddetermine the fluorescent intensity (FI) of the extract.

NOTE: When using FITC-labeled type II collagen as a substrate, the denaturation temperature of intact type II collagen and its TCA fragment
is 41°C and 38°C respectively, higher than those of type I collagen. Therefore, when type II collagen is used as a substrate, increase thedenaturation temperature of collagenase-degradation products from 35°C to 38°C. To shorten the denaturation process when using collagenas a substrate, add a proteinase such as elastase and incubate at 38°C for 20 minutes to digest TCA and TCB fragments into smaller
fragments.

NOTES BEFORE USING ASSAY
NOTE 1: In general, collagenases are secreted as latent proenzymes and require proteolytic conversion for activation. However, collagenase
activity is strictly regulated by tissue inhibitors of metalloproteinase (TIMP) and by 2 macroglobulins (2M) in serum. Thus, different
conditions are required to activate collagenase depending on the experimental purpose, source of enzyme, and levels of proteinase inhibitors
in individual specimens.
NOTE 2: APMA (4-aminophenylmercuric acetate) is widely used to activate latent pro-collagenase (3). Activator 1 (APMA) contains mercury
which is very toxic if inhaled, ingested, or makes contact with skin. Neurological hazard target organs include the kidneys and nerves. Wearsuitable protective gloves, clothing, and eyewear.
NOTE 3: Trypsin activates both latent pro-collagenases and collagenases bound by inhibitors such as 2M and low molecular weightcollagenase inhibitors (4, 5). However, soybean trypsin inhibitor (SBTI) must be added to neutralize the added trypsin before assaying
collagenase activity.
NOTE 4: Potassium thiocyanate (KSCN) or potassium iodide (KI) reactivates collagenases bound by inhibitors such as 2M (6). Anotheractivation method to consider is to dialyze samples against 3M KSCN dissolved in 0.05M Tris-HCl buffer, pH 7.5, at 4°C overnight. Then,remove the KSCN by dialyzing against 0.05M Tris-HCl buffer, pH 7.8, containing 0.2M NaCl and 5mM CaCl2. These reagents may be usefulfor denaturing collagenase inhibitors in sample specimens prior to activating pro-collagenase by APMA (7).
NOTE 5: Both dithiothreitol (DTT) and iodoacetamide have been reported to reactivate collagenases bound by TIMPs (8). However, thesereagents also may inactivate collagenase by reducing the disulfide bonds and alkylating glutamic acid at the active site. Therefore, thelimitations of this method must be taken into consideration.

© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

4ASSAY OUTLINE© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

5

ASSAY PROCEDURE
1.

Prepare Microcentrifuge Tubes: Prepare 1.5 ml microcentrifuge tubes for Buffer, 100% Control, Blank, Reference MMP-8, and Test
Samples as shown on the Collagenase assay sheet at the end of this protocol.
NOTE: Proteins in sample specimens may cause quenching, and consequently, fluorescent intensity (FI) determined in sample tubes
might be underestimated. For example, if the collagenase activity is very low in a sample solution which contains a certain amount ofcontaminant proteins, the FI in the samples will be lower than the Blank value. In order to correct these under-estimated results, theidentical sample mixed with Stop Solution should be added to the Blank tubes and 100% Control tubes. This quenching is mainly causedby turbidity formed by the proteins in the Extraction Buffer. Similarly, colors or dyes in cell culture media also causes quenching. In thiscase, add the same culture medium to Blank and 100% Control tubes.

2.

Add References and Samples: Add the proper amounts of Solution B, reference MMP-8, and test samples to tubes and adjust thefinal volume to 180 µl as shown on the assay sheet. The Buffer tube should only have 380 µl of Solution B.
NOTE: The sample volume may be 5 -180 µl. However, the final volume should be adjusted to 180 µl with Solution B.

3.

Activate collagenase with Activator 1 or Activator 2: To activate collagenase, choose one of the methods described in step 3a OR
3b depending on the experimental purpose, source of collagenase, and enzyme type. In general, it is not necessary to activate
collagenase using both Activator 1 and Activator 2 as the collagenases activated by APMA will be digested and inactivated by trypsin.
Add 10 µl of Activator 1 to the test tubes and incubate for 60 minutes at 35°C. Do not add Activator 1 to undiluted samples because
it is a strong alkaline solution.
ORDissolve one vial of Activator 2 in 1 ml of Solution B. Add 10 µl of Activator 2 to the test tubes and incubate for 60 minutes at 35°C.
NOTE 1: APMA (Activator 1) is recommended as a general activator for latent collagenase activation. However, APMA-activatedcollagenase may be immediately inhibited by proteinase inhibitors coexisting in samples, such as α2M. In these cases, trypsin
activation may be more effective than APMA.
NOTE 2: Trypsin concentration should be optimized for individual samples.
NOTE 3: Collagenase activity is strictly regulated by tissue inhibitors of metalloproteinase (TIMP) and by 2 macroglobulins ( 2M).
For re-activation of inactivated collagenase, Tips for Assaying Collagenase Activity can be obtained from Chondrex, Inc. customer
service (support@chondrex.com)

4.

Add Proteinase Inhibitor: Dissolve one vial of proteinase inhibitor in 1 ml of Solution B. Add 10 µl of proteinase inhibitor into all test
tubes to neutralize non-collagenolytic proteinases in solution.

5.

Prepare 1X FITC: Prepare a 1X FITC-collagen solution by mixing an equal volume of the 2X FITC-collagen and cold Solution A (200µl of the mixture is required for each sample to be tested) in a container protected from light, such as an amber colored tube or bottle(FITC is light sensitive).

6.

Add 1X FITC: Add 200 µl of the 1X FITC-collagen solution into the activated tubes (200 µl). Mix well and incubate at 35°C for 10-120
minutes. Separately incubate the 100% control tube in boiling water for 5 minutes to denature the FITC-collagen.
NOTE: Incubate reference MMP-8 for 60 minutes at 35°C. However, the incubation time for samples will vary depending on thecollagenase activity in sample specimens. Do not incubate test samples longer than 120 minutes otherwise they may yield highbackground levels. Background refers to the degradation of collagen due to extended exposure to high temperatures.

7.

Add Stop Solution: Stop the collagenase reaction by adding 10 µl of Stop Solution to each tube and mix well.

© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

68.

Add Enhancer: Dissolve one vial of Enhancer in 1 ml of Solution B. Add 10 µl of Enhancer to each tube and incubate at 35°C (Cat #3001 Type I collagen) or 38°C (Cat # 3002 Type II collagen) for 10 - 20 minutes. This will further digest the collagenase-degradation
products into smaller fragments.

9.

Add Extraction Buffer: Cool samples to room temperature. Add 400 µl of Extraction Buffer to each tube. Do not use cold buffer. Mix
vigorously and centrifuge at 10,000 rpm for 10 minutes.

10. Transfer: Carefully transfer 200 µl of each supernatant (in duplicate) into the black 96-well plates provided in the kit and determine thefluorescence intensity (FI) at λem = 520 nm and λex = 490 nm.
NOTE 1: Colored samples such as cell culture media may reduce FI approximately 5-10% by quenching, thus the same culture media
must be added to the 100% control tubes for accurate results.
NOTE 2: Supernatants contaminated with pellets in the 96-well plate will lead to high FI values, resulting in overestimated assay results

CALCULATING RESULTS
One unit of collagenolytic activity is defined as the cleavage of 1 µg of collagen per minute (1 unit = 1 µg/minute). Because this kit uses 100µg of collagen per test as a substrate, collagenolytic activity is calculated by the following equation:
FIblank= FI in Blank(FIsample - FIblank) x 100 µg

Collagenase Activity (units/ml):

FIcontrol=FI in 100% Control(FIcontrol - FIblank) x Reaction Time (minute) x Sample Volume (ml)FIsample=FI in Test Samples

NOTE: Reference MMP-8 works to check assay accuracy. Collagenase activities in individual samples must be calculated using the equation
above.

Figure 1a - Standard Curves for Collagenolytic Activity (Type I Collagen Substrate)
6030
Degradation (ug)Degradation (ug)a) 60 min4020b) 2.5 ul
2010000

2.557.5

10Enzyme (ul)0306090Incubation (minutes)a) MMP-8 activity dose response using FITC-Type I collagen as a substrate - Various amounts (2.5 - 10 µl) of reference MMP-8 (100units/ml) were reacted with 100 µg of FITC-Type I collagen at 35°C for 60 minutes. The mixtures were further incubated at 35°C for 10minutes after adding 10 µl of Enhancer.
b) MMP-8 activity time course using FITC-Type I collagen as a substrate - 2.5 µl of MMP-8 (100 units/ml) was reacted with 100 µg FITCType I collagen at 35°C for 90 minutes.

© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

7Figure 1b - Standard Curves for Collagenolytic Activity (Type II Collagen Substrate)
459040a) 60 min

70

Degradation (mg)Degradation (mg)

80

38⁰C

60
50403035⁰C20b) 1.25 m l

35302538 C

20151035⁰C

5100002.557.5Enzym e (ml)0306090

Incubation (minutes)a)

MMP-8 activity dose response using Type II collagen as a substrate - Various amounts (2.5-7.5 µl) of reference MMP-8 (100 units/ml)
were reacted with 100 µg of FITC-labeled type II collagen for 60 minutes at 35°C and 38°C. The mixtures were further incubated at38°C for 20 minutes after adding 10 µl of Enhancer.

b)

MMP-8 activity time course using FITC-Type II collagen as a substrate - 1.25 µl of MMP-8 (100 units/ml) was reacted with 100 µg ofFITC-Type II collagen at 35°C and 38°C for 90 minutes.

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

REFERENCES
1.

K. Terato, R. Hashida, K. Miyamoto, et al., Histological, immunological and biochemical studies on type II collagen-induced arthritis
in rats. Biomedical Res. 3: 495-505 (1982)

2.

K. Terato, Y. Nagai, K. Kawanishi, S. Yamamoto, A rapid assay method of collagenase activity using 14C-labeled soluble collagenas substrate. Biochimica et Biophysica Acta 445: 753-762 (1976)

3.

A. Sellers, E. Cartwright, G. Murphy, J. J. Reynolds, Evidence that latent collagenases are enzyme-inhibitor complexes. J. Biochem.
163: 303-307 (1977)

4.

H. Shinkai, et al., A Complex of Collagenase with Low Molecular Weight Inhibitors in the Culture Medium of Embryonic Chick Skin
Explants. J. Biochem. 81: 261-263 (1977)

5.

H. Shinkai and Y. Nagai, A latent collagenase from embryonic human skin explants. J. Biochem. 81: 1261-1263 (1977)

6.

S. Abe and Y. Nagai, Interaction between tadpole collagenase and human α2-macroglobulin. Biochimica et Biophysica Acta 278:
125-132 (1972)

7.

Y. Murawaki, et al. Serum Collagenase Activity in Patients with Chronic Liver Disease. J. Hepatology 18: 328-334 (1993)

8.

M. R. Rajabi, D. D. Dean, J. F. Woessner, Elevated Tissue Levels of Collagenase During Dilation of Uterine Cervix in HumanParturition. Obstet. Gynecol. 159: 971-976 (1988)© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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

8COLLAGENASE ASSAY SHEETThis assay sheet is provided as a guide. Researchers will need to optimize the assay for their individual needs.
A) Activation
BufferControl(100%)

Blank

Ref 1

Ref 2

Ref 3

Ref 4

Test 1

Test 2

Step 1
Add Reference rhMMP-8
and Test Samples (µl)

000

2.5

5

7.5

10100100

Step 2
Solution B (µl)

380

180

180

177.5

175

172.5

170

8080

Step 3a
Add Activator 1 - APMA (µl)

101010101010101010101010101010OR
Step 3bAdd Activator 2 - Trypsin (µl)

101010Incubate at 35°C for 60 minutes
Step 4
Add Proteinase Inhibitor (µl)

101010101010101010Total Volume (µl)

400200200200200200200200200

BufferControl(100%)

Blank

Ref 1

Ref 2

Ref 3

Ref 4

Test 1

Test 2

Activated Enzyme from Step 4(µl)

400200200200200200200200200

Step 5
Add 1X FITC-Collagen (µl)

0200*

200

200

200200200200200B) Enzyme Assay

React at 35°C (Type I substrate) or 38°C (Type II substrate) for 0-120 minutes
Step 6
Add Stop Solution (µl)

101010101010101010

Step 7
Add Enhancer Solution (µl)

101010101010101010400

400400FI(10)FI(test 1)

FI(test 2)

Incubate at 35°C (Type I substrate) or 38°C (Type II substrate) for 10-20 minutes
Step 8Add Extraction Buffer (µl)

400

400

400

400

400

400

Mix well and centrifuge at 10,000 rpm for 10 minutes.
Transfer 200 µl of supernatant into a 96-well flat bottom black plate
Step 9Determine FI at Em 520/Ex 490

FIblank

FIcontrol =100 µg
collagenFI(0)FI(2.5)FI(5)FI(7.5)

Calculate collagenase activity by comparing FItest and FIcontrol.
*Heat denatured 1X FITC-collagen© 2022 Chondrex, Inc. All Rights Reserved, 3001, 3002 6.2
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