Type I Collagenase Activity Assay Kit - Millipore

Type I Collagenase Activity
Assay Kit
Cat. No. ECM710
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
USA & Canada
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Introduction
Collagenases (MMP-1, MMP-8, and MMP-13) are members of a family of
matrix metalloproteinases (MMPs) which are able to degrade components of the
extracellular matrix.
The CHEMICON ® MMP Collagenase Activity Assay Kit provides a quick,
efficient and sensitive system for evaluation of collagenase activity in serum,
cell lysates, supernatants, and other samples, or collagenase inhibitor screening.
The 96-well format of the kit is significantly faster than traditional zymography
for evaluation of multiple samples, offers the versatility of 8 x 12 strip well
design, can be easily measured with a microplate reader, and contains purified
Human MMP-1 as a positive control. Assay time is approximately 4 hours.
Test Principle
The CHEMICON ® MMP Collagenase Activity Assay Kit utilizes Bovine
biotinylated collagen as a substrate. This native, triple helical substrate is
readily cleaved by active MMP-1, MMP-8 and MMP-13 (collagenotic)
enzymes. Following addition of the enhancer, the remaining biotinylated
fragments are then transferred to a biotin-binding 96-well plate and detected
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with streptavidin-enzyme complex. Addition of enzyme substrate results in a
colored product, detectable by its Optical Density at 450 nm (OD450).
Application
The CHEMICON ® MMP Collagenase Activity Assay Kit is ideal for
measurement of collagenase activity or collagenase inhibitor potency in culture
supernatants, serum, cell lysates or other collagenase-containing samples.
According to literature values, collagenase levels for conditioned media of
cultured cells and serum are typically in the range of 10-200 ng/mL. The
CHEMICON ® MMP Collagenase Activity Assay Kit is a quantitative assay.
Experimental studies using purified, APMA-activated MMP-1 have established
the analytical sensitivity of the kit to be less than 5 ng of MMP per mL, which is
much more sensitive than traditional SDS-PAGE/densitometry.
APMA (p-Aminophenylmercuric Acetate)-activated MMP-1 is supplied as a
positive control for quantitative purposes. Recombinant MMP-8 and MMP-13
standards for quantitative purposes are available separately as catalog numbers
CC067 and CC1047, respectively. Customers have the option of measuring
endogenous MMP activity levels in their samples or activating their samples
with APMA prior to the assay.
Each CHEMICON ® MMP Collagenase Activity Assay Kit contains sufficient
reagents for the evaluation of 96 samples, including positive control. Duplicate
or triplicate samples are suggested.
The CHEMICON ® MMP Collagenase Activity Assay Kit is intended for
research use only; not for diagnostic or therapeutic applications.
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Kit Components
1. MMP-1 Positive Control: (Part No. 90054) One lyophilized vial containing
500 ng of APMA-activated human MMP-1.
2. Biotinylated Collagenase Substrate: (Part No. 90052) One 600 µL vial.
3. Sample Diluent, 5X: (Part No. 90056) One 10 mL bottle.
4. Biotin-binding Plate: (Part No. 90023) One 96-well biotin-binding plate.
5. Enhancer: (Part No. 90055) One lyophilized vial.
6. Streptavidin-Enzyme Conjugate: (Part No. 90053) One 21 µL vial.
7. Substrate Solution: (Part No. 90028) One 12 mL vial.
8. Stop Solution: (Part. No. 60193) One 12 mL vial.
9. Assay Buffer, 10X: (Part. No. 90027) One 100 mL bottle.
Materials Not Supplied
1. APMA for MMP activation of samples (if desired)
2. Multichannel pipette and tips
3. Microplate reader (450 nm)
4. 37°C incubator
5. Clean 96-well microtiter plate for performing incubations.
6. Deionized water
Special Precautions
1. Maintain all kit components at 2° to 8°C. The MMP-1 Positive Control and
Biotinylated Collagenase Substrate are especially temperature sensitive, and
mishandling may negatively effect the kit’s performance.
2. Due to the fragile, native structure of the Biotinylated Collagenase
Substrate, perform all dilutions and handling of this material at 2° to 8°C.
Do not freeze.
3. Do not add the Biotinylated Collagenase Substrate directly to the 5X
Sample Diluent. This will denature the substrate.
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4. Assay incubations must be performed at 37±1°C for optimal enzymatic
activity, while maintaining the substrate’s native structure. Excessive heat
will denature the substrate.
Sample Preparation/Activation
1. Prepare Collagenase-containing samples as desired. MMP-1 Positive
Control has been pre-activated with APMA and does not require further
activation. 10-100 µL of diluted samples will be required per well
(triplicate sample wells are recommended). Note: Samples should not
contain EDTA, EGTA or other chelators of divalent cations.
2. If APMA activation of samples is desired, prepare a 20mM APMA
solution.
a) Dissolve 3.5 mg of APMA (not provided) in 0.5 mL of 0.1N NaOH.
This stock solution is stable for approximately 3 days at 4°C.
3. Activate samples by adding 350 µL of sample to 100 µL of 250 mM Tris,
pH 7.5, 50 mM CaCl2, plus 50 µL of APMA solution from Step 2.
4. Incubate sample for 5 hours at 37°C. Use activated collagenase within one
day for best results.
Generating a Standard Curve with the MMP-1 Positive Control
1. Rehydrate the vial of MMP-1 Positive Control with 100 µL of deionized
water and mix well. Use immediately or aliquot and freeze at -80°C for
longer storage. Avoid repeated freeze/thaw cycles.
Note: Once reconstituted, the MMP-1 Positive Control concentration will
be 5 ng/µL.
2. In a clean, 96-well microtiter plate, serially dilute the MMP-1 Positive
Control 1:2 by the following method:
• Add 20 µL of 1X Sample Diluent to wells B1-H1.
• Add 40 µL of reconstituted MMP-1 Positive Control (step 1) to
well A1.
• Transfer 20 µL of MMP-1 Positive Control (starting from well A1)
into the next well already containing 20 µL of 1X Sample Diluent,
as illustrated below in Template #1 until the MMP-1 has been
diluted 64-fold (2 6 ) in row G. Leave row H as a zero control.
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Template #1: Dilution of MMP-1 Positive Control
A
1 2 3 4 5 6 7 8 9 10 11 12
neat
(5 ng/µL)
B 1:2
C 1:4
D 1:8
E 1:16
F 1:32
G 1:64
H 0
• Maintaining the same pattern, on the clean part of the plate transfer
10 µL of the diluted MMP-1 positive control (wells A1-H1). See
template #2 for the respective amount of MMP-1 transferred per
well.
Template #2: Transfer of diluted MMP-1 Positive Control (10 µL)
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
5
neat
(50 ng)
1:2
(25 ng)
1:4
(12.5 ng)
1:8
(6.25 ng)
1:16
(3.13 ng)
1:32
(1.56 ng)
1:64
(0.78 ng)
0
(0 ng)
3. Proceed with the assay from step 2 of “Assay Instructions” using 10 µL as
the sample volume.

Assay Instructions
1. First, determine whether 10 µL, 50 µL, or 100 µL Test Samples will be
used. The CHEMICON ® MMP Collagenase Activity Assay Kit can
accurately detect collagenase activity with analytical sensitivity to less than
10 ng/mL. For samples with low expected collagenase content (less than
20 ng/mL) a 100 µL sample size is suggested. For samples with higher
activity, a 10 µL sample is recommended.
2. In a small vial, pre-dilute the appropriate volume of Biotinylated
Collagenase Substrate required for the number of wells to be assayed with
cold, deionized water. Mix well and store at 2° to 8°C until needed.
Depending on the sample volume, the final pre-diluted substrate volume
will vary, select the appropriate chart below.
For 10 µL Samples
Number of wells to be
Assayed
Volume of Biotinylated
Collagenase Substrate
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Volume of cold,
deionized water
96 500 µL 9.500 mL
48 250 µL 4.750 mL
24 125 µL 2.375 mL
16 83.3 µL 1.583 mL
8 41.6 µL 0.792 mL
For 50 µL Samples
Number of wells to be
Assayed
Volume of Biotinylated
Collagenase Substrate
Volume of cold,
deionized water
96 500 µL 5.500 mL
48 250 µL 2.750 mL
24 125 µL 1.375 mL
16 83.3 µL 0.916 mL
8 41.6 µL 0.458 mL

For 100 µL Samples
Number of wells to be
Assayed
Volume of Biotinylated
Collagenase Substrate
96 500 µL 500 µL
48 250 µL 250 µL
24 125 µL 125 µL
16 83.3 µL 83.3 µL
8 41.6 µL 41.6 µL
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Volume of cold,
deionized water
Note: Once diluted, Biotinylated Collagenase Substrate should be used
immediately. This material should not be stored in the diluted form and should
not be frozen.
3. Rehydrate the vial of Enhancer with 1.1 mL of deionized water. Mix well
and store at 2° to 8°C until needed.
4. For 10 µL samples: In a clean, 96-well microtiter plate (triplicate is
recommended), add 10µL of MMP test sample (prepared as described in
“Sample Preparation”) to each well, followed by 30 µL of 5X Sample
Diluent. With the diluent and sample added, then add 100 µL of diluted
Biotinylated Collagenase substrate from Step 2. The total volume should
be 140 µL.
For 50 µL samples: In a clean, 96-well microtiter plate (triplicate is
recommended), add 50µL of MMP test sample (prepared as described in
“Sample Preparation”) to each well, followed by 30 µL of 5X Sample
Diluent. With the diluent and sample added, then add 60 µL of diluted
Biotinylated Collagenase substrate from Step 2. The total volume should
be 140 µL.
For 100 µL samples: In a clean, 96-well microtiter plate (triplicate is
recommended), add 100µL of MMP test sample (prepared as described in
“Sample Preparation”) to each well, followed by 30 µL of 5X Sample
Diluent. With the diluent and sample added, then add 10 µL of diluted
Biotinylated Collagenase substrate from Step 2. The total volume should
be 140 µL.
5. Cover the plate and incubate for 2 hours at 37°C.
6. Add 10 µL of Enhancer (from step 3) to each well.
7. Cover the plate and incubate for an additional 30 minutes at 37°C.

8. Rehydrate Biotin-binding Plate with ~200 µL of PBS per well and let the
plate stand at room temperature for 1 minute. Do not use Assay Buffer for
rehydration.
9. Aspirate PBS from Biotin-binding Plate, being careful not to scratch the
plate.
10. Transfer 100 µL of the Sample/Biotinylated Collagenase Substrate mixture
from Step 7 to the rehydrated Biotin-binding Plate and incubate for 30
minutes at 37°C.
11. Dilute sufficient 10X Assay Buffer with deionized water for the number of
samples to be assayed. If using the entire 96-well plate, add the entire
100 mL volume of 10X Assay Buffer to 900 mL deionized water. If using
fewer wells, scale down amount of Assay Buffer and deionized water
accordingly.
12. Wash 5 x with 200 µL/well of diluted Assay Buffer.
13. Prepare a 1:3000 dilution of Streptavidin-Enzyme Conjugate in Assay
Buffer by adding 4 µL of Streptavidin-Enzyme Conjugate to 12 mL of
Assay Buffer.
14. Add 100 µL of diluted Streptavidin-Enzyme Conjugate to each well and
incubate for 30 minutes at 37°C.
15. Wash 5 x with 200 µL of diluted Assay Buffer per well.
16. Add 100 µL of Substrate Solution and incubate at room temperature for 5 to
20 minutes.
17. Positive control wells should develop faint color. Stop reaction by adding
100 µL of Stop Solution to each well.
18. Measure Optical Density at 450 nm on a microplate reader to compare the
activity of the test samples.
Storage
Store kit materials at 2° to 8°C up to their expiration date. Do not freeze.
Calculation of Results
Optical Density values obtained with the CHEMICON ® MMP Collagenase
Activity Assay Kit may be compared with the provided MMP-1 standard for
quantitation.
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The following charts illustrate typical results upon dilution of the MMP Positive
Control contained in the kit. One should use the data below for reference only.
This data should not be used to interpret actual assay results.
OD 450nm
OD 450nm
2.000
1.750
1.500
1.250
1.000
0.750
0.500
0.250
0.000
2.000
1.750
1.500
1.250
1.000
0.750
0.500
0.250
0.000
MMP-1 Collagenase Activity Assay
0.78125
1.5625
9
3.125
0 1 10 100
6.25
12.5
Active MMP-1 Standard (ng)
MMP-8 and MMP-13 Collagenase Activity Assays
0 1 10 100
Active Collagenase (ng)
25
50
MMP-8
MMP-13
MMP-2

References
For the latest listing of product citations, please see the references tab at
ECM700 at www.chemicon.com.
Warranty
These products are warranted to perform as described in their labeling and in
CHEMICON ® literature when used in accordance with their instructions.
THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS
EXPRESSED WARRANTY AND CHEMICON ® DISCLAIMS ANY
IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF
FITNESS FOR PARTICULAR PURPOSE. CHEMICON ® ’s sole obligation
and purchaser’s exclusive remedy for breach of this warranty shall be, at the
option of CHEMICON ® , to repair or replace the products. In no event shall
CHEMICON ® be liable for any proximate, incidental or consequential damages
in connection with the products.
©2000, 2004: CHEMICON ® International, Inc. - By CHEMICON ®
International, Inc. All rights reserved. No part of these works may be
reproduced in any form without permissions in writing.
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Cat. No. ECM710
August 2005
Revision F: 41072