ab196999
Collagenase Activity
Assay Kit (Colorimetric)
Instructions for Use
For the rapid, sensitive and accurate measurement of collagenase
activity in purified collagenase and bacterial extract.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 7 December 2015
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
4
4
5
5
6
7
ASSAY PREPARATION
9.
10.
REAGENT PREPARATION
SAMPLE PREPARATION
8
9
ASSAY PROCEDURE and DETECTION
11.
ASSAY PROCEDURE and DETECTION
10
DATA ANALYSIS
12.
13.
CALCULATIONS
TYPICAL DATA
12
13
RESOURCES
14.
15.
16.
17.
18.
QUICK ASSAY PROCEDURE
TROUBLESHOOTING
FAQ
INTERFERENCES
NOTES
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16
18
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1
INTRODUCTION
1.
BACKGROUND
Collagenase Activity Assay Kit (colorimetric) (ab196999) provides a
quick and easy way to determine activity of collagenase. This assay
measures collagenase activity using a synthetic peptide (FALGPA) that
mimics the structure of collagen. It is suitable for measuring activity of
bacterial collagenases such as from Clostridium histolyticum type I-XI.
In addition, it can also be used to screen/characterize collagenase
inhibitors. The limit of detection for this assay is 0.02 mU collagenase.
Collagenase is an enzyme in the matrix metalloproteinase family that
breaks down collagen, assisting in degradation of the extracellular
matrix, which is a key step in the pathogenesis of bacteria. Collagen is
an abundant structural protein present in the connective tissue of
animals. Collagenase has been used clinically for the treatment of
Dupuytren’s contracture, an affliction characterized by a thickening of
connective tissue.
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2
INTRODUCTION
2. ASSAY SUMMARY
Sample preparation
Set up reaction wells or inhibitor screening wells
Add reaction mix
Measure optical density (OD345 nm) in a kinetic mode
at 37°C for 5 – 15 min*
*For kinetic mode detection, incubation time given in this summary is
for guidance only.
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3
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at -20ºC in the dark immediately upon receipt. Kit has a
storage time of 1 year from receipt, providing components have
not been reconstituted.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in section 5.
Aliquot components in working volumes before storing at the
recommended temperature. Reconstituted components are stable
for 2 months.
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4
GENERAL INFORMATION
5. MATERIALS SUPPLIED
Collagenase Assay Buffer
20 mL
Storage
Condition
(Before
Preparation)
-20°C
Collagenase (0.35 U/mL)
1 mL
-20°C
-20°C
Collagenase Substrate (FALGPA)
1 mL
-20°C
-20°C
Inhibitor (1,10-Phenanthroline) 1M
50 µL
-20°C
-20°C
Item
Amount
Storage
Condition
(After
Preparation)
4°C / -20°C
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully perform this assay:

MilliQ water or other type of double distilled water (ddH2O)

PBS

HBSS (Hank’s Balanced Salt Solution)

Microcentrifuge

Pipettes and pipette tips

Colorimetric microplate reader – equipped with filter for OD=345
nm

96 well plate: clear plates for colorimetric assay

Heat block or water bath
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5
GENERAL INFORMATION
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not use kit or components if it has exceeded the expiration date
on the kit labels.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
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6
GENERAL INFORMATION
8. TECHNICAL HINTS

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff with
any questions.

Keep enzymes and heat labile components and samples on ice
during the assay.

Make sure all buffers and developing solutions are at room
temperature before starting the experiment.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Avoid foaming
components.

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers.

Ensure plates are properly sealed or covered during incubation
steps.

Ensure complete removal of all solutions and buffers from tubes or
plates during wash steps.

Make sure you have the appropriate type of plate for the detection
method of choice.

Make sure the heat block/water bath and microplate reader are
switched on before starting the experiment.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION

Briefly centrifuge small vials at low speed prior to opening.
9.1
Collagenase Assay Buffer:
Ready to use as supplied. Equilibrate to room temperature
before use. Store at -20°C.
9.2
Collagenase Positive Control:
Ready to use as supplied. Aliquot positive control so that
you have enough volume to perform the desired number of
tests. Store at -20°C. Avoid repeated freeze/thaw cycles.
Use within 2 months. Keep on ice while in use.
9.3
Collagenase Substrate (FALGPA):
Ready to use as supplied. Aliquot substrate so that you have
enough volume to perform the desired number of tests.
Store at -20°C. Avoid repeated freeze/thaw cycles. Use
within 2 months. Keep on ice while in use.
9.4
Inhibitor:
Ready to use as supplied. Aliquot inhibitor so that you have
enough volume to perform the desired number of tests.
Store at -20°C. Avoid repeated freeze/thaw cycles. Use
within 2 months. Keep on ice while in use.
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8
ASSAY PRE
ASSAY PREPARATION
10.SAMPLE PREPARATION
General Sample information:

We recommend performing several dilutions of your sample to
ensure the readings are within the standard value range.

We recommend that you use fresh samples. If you cannot perform
the assay at the same time, we suggest that you complete the
Sample Preparation step before storing the samples. Alternatively,
if that is not possible, we suggest that you snap freeze cells or
tissue in liquid nitrogen upon extraction and store the samples
immediately at -80°C. When you are ready to test your samples,
thaw them on ice. Be aware however that this might affect the
stability of your samples and the readings can be lower than
expected.
11.1
Purified collagenase:
Dissolve test collagenase in cold ddH2O or HBSS.
Suggested range for collagenase testing = 0.02 – 10 mU.
11.2
Bacterial extracts:
Lyse bacterial cells in cold PBS.
We suggest performing several dilutions of the sample to find
the optimal values to perform the assay.
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ASSAY PROCEDURE and DETECTION
11.ASSAY PROCEDURE and DETECTION
●
Equilibrate all materials and prepared reagents to room
temperature prior to use.
●
It is recommended to assay all controls and samples in
duplicate.
11.1
Set up Reaction wells:
Prepare reaction wells following set up in the table below:
Component
Test
collagenase
sample
Collagenase
0.35 U/mL
Inhibitor
Assay Buffer
11.2
Test well
(µL)
Positive
control
well (µL)
Inhibitor
well (µL)
Background
well (µL)
2 - 10
0
0
0
0
10
10
0
0
0
2
0
Up to 100
Up to 100
Up to 100
100
Inhibitor Screening (optional):
-
If collagenase inhibitors are to be tested, dissolve test
collagenase inhibitor to 100X in an appropriate solvent.
-
Then set up wells as follows:
Component
Test collagenase
inhibitor
Collagenase
0.35 U/mL
Solvent
Assay Buffer
Test well
(µL)
Enzyme
control well
(µL)
Solvent
control well
(µL)
2
0
0
10
10
10
0
0
2
Up to 100
Up to 100
Up to 100
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ASSAY PRE
ASSAY PROCEDURE and DETECTION
11.3
Reaction Mix:
Prepare 100 µL of Reaction Mix for each reaction
Component
Reaction Mix (µL)
Collagenase Substrate
40
Collagenase Assay Buffer
60
Mix enough reagents for the number of assays (test
collagenase, positive control, inhibitor control, background
controls, test inhibitor and enzyme control) to be performed.
Prepare a master mix of the Reaction Mix to ensure
consistency. We recommend the following calculation:
X µL component x (Number samples + controls +1).
11.4 Add 100 µL of Reaction Mix into all wells and mix.
11.5
Measure absorbance immediately on a microplate reader at
OD = 345 nm in a kinetic mode, every 2 -3 minutes, for 5 –
15 minutes.
Low activity samples can be measured for 1 – 3 hours.
High activity samples will consume substrate within 3
minutes. Dilute enzyme and re-measure if necessary
NOTE: High activity samples will consume substrate within 3
minutes. Dilute enzyme and measure again if necessary.
We recommend measuring the OD in kinetic mode, and
choosing two time points (T1 and T2) in the linear portion of the
time course to calculate the collagenase activity.
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11
DATA ANALYSIS
12.CALCULATIONS

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
12.1
Average the duplicate reading for each standard and
sample.
12.2
Take the absorbance (A345nm1 and A345nm2) at two time
points (T1 and T2) in the linear range. There should be at
least two readings in between and at least 1 minute apart.
12.3
To determine activity, use the following equation:
𝑆𝑎𝑚𝑝𝑙𝑒 𝐶𝑜𝑙𝑙𝑎𝑔𝑒𝑛𝑎𝑠𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦
‒ ∆𝐴
(
∆𝑇
=
345𝑛𝑚
𝑇𝑒𝑠𝑡 ‒
‒ ∆𝐴345𝑛𝑚
𝑅𝑒𝑎𝑔𝑒𝑛𝑡 𝐵𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑 𝑥 0.2 𝑥 𝐷𝐹
∆𝑇
𝑈/𝑚𝐿
0.53 × 𝑉
)
Where:
∆A345nm = Difference between A345nm2 and A345nm1
∆T = Difference between T2 and T1
0.2 = Reaction volume (mL)
DF = Dilution Factor
0.53 = millimolar extinction coefficient of collagenase substrate
V = Enzyme volume (mL)
12.4
For inhibitor screen, calculate percent inhibition using the
following equation::
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐸𝑛𝑧𝑦𝑚𝑒) ‒ 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟)
% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =
× 100
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐸𝑛𝑧𝑦𝑚𝑒)
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12
DATA ANALYSIS
13.TYPICAL DATA
Figure 1. Collagenase activity over 10 minutes.
Figure 2: Enzyme activity of collagenase and inhibition by 10 mM (1,10)Phenanthroline.
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13
RESOURCES
14.QUICK ASSAY PROCEDURE
NOTE: This procedure is provided as a quick reference for
experienced users. Follow the detailed procedure when performing
the assay for the first time.

Prepare collagenase, inhibitor, and assay buffer; (aliquot if
necessary); get equipment ready.

Prepare samples in duplicate.

Set up plate as follows:
Component
Test
collagenase
sample
Collagenase
0.35 U/mL
Inhibitor
Assay
Buffer

Test well
(µL)
Positive
control
well (µL)
Inhibitor
well
(µL)
Background
well ((µL)
2 - 10
0
0
0
10
10
10
0
0
Adjust
volume
to 100
0
Adjust
volume to
100
2
Adjust
volume
to 100
0
100
If collagenase inhibitors are being tested (optional): prepare 100X
concentrated test collagenase inhibitors and set up wells as
follows:
Component
Test
collagenase
sample
Collagenase
0.35 U/mL
Solvent
Assay
Buffer
Test well
(µL)
Enzyme
control well
(µL)
Solvent
control well
(µL)
2
0
0
10
10
10
0
Adjust
volume to
100
0
2
Adjust
volume to 100
Adjust
volume to 100
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RESOURCES

Prepare Reaction Mix (Number samples + controls + 1).
Component
Collagenase Substrate
Colorimetric Reaction
Mix (µL)
40
Collagenase Assay Buffer
60

Add 100 µL of Reaction Mix to all wells.

Immediately measure output (A1) on a microplate reader at time
(T1), at OD345 nm.

Incubate at 37°C for 5 – 15 minutes (or 1 -3 hours for low activity
samples).

Measure output (A2) on a microplate reader at time (T2), at
OD345 nm.
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RESOURCES
15.TROUBLESHOOTING
Problem
Assay not
working
Sample with
erratic
readings
Lower/
Higher
readings in
samples and
Standards
Cause
Solution
Use of ice-cold buffer
Buffers must be at room
temperature
Plate read at
incorrect wavelength
Check the wavelength and filter
settings of instrument
Use of a different 96well plate
Colorimetric: Clear plates
Fluorometric: black wells/clear
bottom plate
Samples not
deproteinized (if
indicated on protocol)
Cells/tissue samples
not homogenized
completely
Samples used after
multiple free/ thaw
cycles
Use of old or
inappropriately stored
samples
Presence of
interfering substance
in the sample
Use PCA precipitation protocol for
deproteinization
Use Dounce homogenizer,
increase number of strokes
Aliquot and freeze samples if
needed to use multiple times
Use fresh samples or store at 80°C (after snap freeze in liquid
nitrogen) till use
Check protocol for interfering
substances; deproteinize samples
Improperly thawed
components
Thaw all components completely
and mix gently before use
Allowing reagents to
sit for extended times
on ice
Always thaw and prepare fresh
reaction mix before use
Incorrect incubation
times or temperatures
Verify correct incubation times
and temperatures in protocol
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RESOURCES
Problem
Standard
readings do
not follow a
linear pattern
Unanticipated
results
Cause
Solution
Pipetting errors in
standard or reaction
mix
Avoid pipetting small volumes
(< 5 µL) and prepare a master mix
whenever possible
Air bubbles formed in
well
Pipette gently against the wall of
the tubes
Standard stock is at
incorrect
concentration
Always refer to dilutions on
protocol
Measured at incorrect
wavelength
Check equipment and filter setting
Samples contain
interfering
substances
Sample readings
above/ below the
linear range
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Troubleshoot if it interferes with
the kit
Concentrate/ Dilute sample so it is
within the linear range
17
RESOURCES
16.FAQ
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RESOURCES
17.INTERFERENCES
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RESOURCES
18.NOTES
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RESOURCES
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RESOURCES
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RESOURCES
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