ab196999 Assay Kit (Colorimetric) Collagenase Activity · A ‘test’ simply refers to a single...

Version 1 Last Updated 18 March 2015

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.

ab196999Collagenase Activity Assay Kit (Colorimetric)

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Table of Contents

INTRODUCTION1. BACKGROUND 22. ASSAY SUMMARY 3

GENERAL INFORMATION3. PRECAUTIONS 44. STORAGE AND STABILITY 45. MATERIALS SUPPLIED 56. MATERIALS REQUIRED, NOT SUPPLIED 57. LIMITATIONS 68. TECHNICAL HINTS 7

ASSAY PREPARATION9. REAGENT PREPARATION 810. SAMPLE PREPARATION 9

ASSAY PROCEDURE and DETECTION11. ASSAY PROCEDURE and DETECTION 10

DATA ANALYSIS12. CALCULATIONS 1213. TYPICAL DATA 13

RESOURCES14. QUICK ASSAY PROCEDURE 1415. TROUBLESHOOTING 1616. FAQ 1817. INTERFERENCES 1918. NOTES 20


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.


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.


GENERAL INFORMATION

3. PRECAUTIONSPlease 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 STABILITYStore 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.


GENERAL INFORMATION

5. MATERIALS SUPPLIED

Item AmountStorage

Condition(Before

Preparation)

StorageCondition

(After Preparation)

Collagenase Assay Buffer 20 mL -20°C 4°C / -20°CCollagenase (0.35 U/mL) 1 mL -20°C -20°CCollagenase Substrate (FALGPA) 1 mL -20°C -20°CInhibitor (1,10-Phenanthroline) 1M 50 µL -20°C -20°C

6. MATERIALS REQUIRED, NOT SUPPLIEDThese 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


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.


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 or bubbles when mixing or reconstituting 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.


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.

ASSAY PRE


ASSAY PREPARATION

10.SAMPLE PREPARATIONGeneral 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.


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:

11.2 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 well (µL)

Positive control

well (µL)Inhibitor well (µL)

Background well (µL)

Test collagenase sample

2 - 10 0 0 0

Collagenase 0.35 U/mL 10 10 10 0

Inhibitor 0 0 2 0Assay Buffer Up to 100 Up to 100 Up to 100 100


Enzyme control well

(µL)

Solvent control well

(µL)Test collagenase inhibitor 2 0 0

Collagenase 0.35 U/mL 10 10 10

Solvent 0 0 2Assay Buffer Up to 100 Up to 100 Up to 100

ASSAY PRE


ASSAY PROCEDURE and DETECTION

11.3 Reaction Mix:Prepare 100 µL of Reaction Mix for each reaction

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.

Component Reaction Mix (µL)

Collagenase Substrate 40Collagenase Assay Buffer 60


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 = ( ‒ ∆𝐴345𝑛𝑚∆𝑇 𝑇𝑒𝑠𝑡 ‒

‒ ∆𝐴345𝑛𝑚∆𝑇 𝑅𝑒𝑎𝑔𝑒𝑛𝑡 𝐵𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑)𝑥 0.2 𝑥 𝐷𝐹

0.53 × 𝑉 𝑈/𝑚𝐿

Where: ∆A345nm = Difference between A345nm2 and A345nm1

∆T = Difference between T2 and T1

0.2 = Reaction volume (mL) DF = Dilution Factor0.53 = millimolar extinction coefficient of collagenase substrateV = Enzyme volume (mL)

12.4 For inhibitor screen, calculate percent inhibition using the following equation::

% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐸𝑛𝑧𝑦𝑚𝑒) ‒ 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟)

𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦(𝐸𝑛𝑧𝑦𝑚𝑒)× 100


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.


RESOURCES

14.QUICK ASSAY PROCEDURENOTE: 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:


Positive control

well (µL)

Inhibitor well (µL)

Background well ((µL)

Test collagenase sample

2 - 10 0 0 0

Collagenase 0.35 U/mL 10 10 10 0

Inhibitor 0 0 2 0

Assay Buffer

Adjust volume to 100

Adjust volume to

100

Adjust volume to 100

100

If collagenase inhibitors are being tested (optional): prepare 100X concentrated test collagenase inhibitors and set up wells as follows:


Enzyme control well

(µL)

Solvent control well

(µL)Test collagenase sample

2 0 0

Collagenase 0.35 U/mL 10 10 10

Solvent 0 0 2

Assay Buffer

Adjust volume to

100Adjust

volume to 100Adjust

volume to 100


RESOURCES

Prepare Reaction Mix (Number samples + controls + 1).

Component Colorimetric Reaction Mix (µL)

Collagenase Substrate 40Collagenase 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.


RESOURCES

15.TROUBLESHOOTING

Problem 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

Assay not

workingUse of a different 96-

well plate

Colorimetric: Clear platesFluorometric: black wells/clear

bottom plateSamples not

deproteinized (if indicated on protocol)

Use PCA precipitation protocol for deproteinization

Cells/tissue samples not homogenized

completely

Use Dounce homogenizer, increase number of strokes

Samples used after multiple free/ thaw

cycles

Aliquot and freeze samples if needed to use multiple times

Use of old or inappropriately stored

samples

Use fresh samples or store at - 80°C (after snap freeze in liquid

nitrogen) till use

Sample with erratic readings

Presence of interfering substance

in the sample

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

Lower/ Higher readings in samples and Standards Incorrect incubation

times or temperaturesVerify correct incubation times and temperatures in protocol


RESOURCES

Problem Cause SolutionPipetting errors in

standard or reaction mix

Avoid pipetting small volumes (< 5 µL) and prepare a master mix

whenever possibleAir bubbles formed in

wellPipette gently against the wall of

the tubes

Standard readings do not follow a linear pattern 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

Troubleshoot if it interferes with the kit

Unanticipated results

Sample readings above/ below the

linear range

Concentrate/ Dilute sample so it is within the linear range


RESOURCES

16.FAQ


RESOURCES

17. INTERFERENCES


RESOURCES

18.NOTES


RESOURCES

RESOURCES 23

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ab196999 Assay Kit (Colorimetric) Collagenase Activity · A ‘test’ simply refers to a single...

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