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Please contact your local representative.", "ReadyForUse": "", "OrderInformation": "Unit of measure is \"piece\"." } ] }, "Product2Products": { "Product2ProductReference": [ ] }, "ProductSpec": [ { "ProductSpecVariant": { "Chapters": [ { "Language": "en", "Value": "Store the kit at -15 to -25°C.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "
Conversion Factors from Wünsch Units to FALGPA Units
\n\n\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n
EnzymeFactor
Collagenase I           9.1
Collagenase II2.7
Liberase Blends3.0
", "Country": "XG", "Code": "Unit Conversion", "Name": "Unit Conversion" }, { "Language": "en", "Value": "A quantitative, functional assay is performed to determine the lot-specific activities of Calibrators A to E and recover the activity of an internal standard.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "Standard Laboratory Equipment\n\n
    \n\t
  • Polypropylene tubes for preparation of sample dilutions
  • \n\t
  • 0.5 to 10 μl and 10 to 100 μl micropipettes
  • \n\t
  • 96-well transparent flat-bottom microplate treated for cell culture from the following list or equal product: \n\t
      \n\t\t
    • Thermo Scientific Nunclon Delta, 167314 
    • \n\t\t
    • Corning Falcon TC-treated, 353075
    • \n\t\t
    • Corning Costar TC-treated, 3997 
    • \n\t\t
    • Charles River Endosafe, M9005
    • \n\t\t
    • Greiner Cellstar, 655180
    • \n\t
    \n\t
  • \n\t
  • Multiwell plate reader capable of determining initial maximal rates (Vmax) of absorbance at 340 nm with at least two data points per well per minute
  • \n
\n
\nStandard Laboratory Reagents\n\n
    \n\t
  • Purified water for the blank
  • \n\t
  • Diluent for preparation of sample dilutions
  • \n
\n
\nFor Positive Control\n\n
    \n\t
  • Liberase Blend (see section Ordering Information)
  • \n
", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "Total assay time: approximately 30 minutes.", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n
ObservationPossible causeRecommendation
Activity of samples too low. Low concentration after dilution of highly concentrated samples.Reduce dilution factor for highly concentrated samples.
Activity of samples too high.Activity exceeds measuring range.Dilute samples to ensure that they are within the specified measuring range.
Background of sample matrix.Check analyte-free sample matrix for background activity value (negative control). Correct sample values for background activity of matrix if the background Vmax value of the negative control is higher than the Vmax value of Calibrator A.
Contamination of samples with Liberase Blend from lab workspace.Repeat the experiment in a different workspace using a new kit. Be careful when reconstituting lyophilizates. Thoroughly
\n\t\t\tclean contaminated workspace.
Variations too highInsufficient mixing of calibrator or sample and FALGPA Solution.Ensure complete mixing by pipetting up and down several times and/or including an agitation/shaking step in the multiwell plate reader software.
Cross-contamination between wells during the agitation/shaking step.Ensure that the agitation/shaking step is performed at the lowest possible setting on the plate reader.
Unsuitable microplate.Use transparent flat-bottom 96-well plates.
Sample matrix difficult to pipette due to viscosity or composition.Carefully pipette samples without droplets on outside of tip. Avoid high aspiration or ejection velocity when pipetting the samples.
Pipetting precision is not optimal.Check the precision of the pipetting.
Determination of Vmax values disturbed by air bubbles or other artefacts.Check the measured absorbance rates in the instrument software. Make sure Vmax values are determined in a sufficiently linear phase near the start of the enzymatic reaction. Vary the measurement time range and/or increase the number of data points to improve linearity. The time between sample pipetting and measuring should be minimized to achieve the highest accuracy and precision. If the high variations persist, repeat the test run and avoid/remove air bubbles in the wells by carefully agitating the microplate.
", "Country": "XG", "Code": "Troubleshooting", "Name": "Troubleshooting" }, { "Language": "en", "Value": "How this Product Works
\nThe product is a test for the quantitative determination of the enzymatic activity of Collagenase I, Collagenase II, and blends thereof (Roche Diagnostics Liberase Blends), and is based on the principle of a continuous hydrolytic reaction of a colorimetric peptide substrate.
\n
\n                   Collagenase
\nFALGPA  ---------------------> FAL + GPA
\n
\nFALGPA = N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala
\nFAL = N-(3-[2-Furyl]acryloyl)-Leu
\nGPA = Gly-Pro-Ala
\n
\nThe hydrolysis of FALGPA is observed as a decrease in optical absorbance at 340 nm, recorded as continuous absorbance rate, and expressed as enzymatic units, where one unit of Collagenase activity is defined as hydrolysis of 1.0 μmole of FALGPA per minute at +25°C and pH 8.8 in the presence of calcium ions (Van Wart, H.E., et al., 1981).
\nThe format is a photometric assay using a 96-well plate and is applicable with most commercially available flat-bottom plates and plate readers.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "
All solutions supplied with the kit are ready to use once thawed and centrifuged. Do not further dilute the Calibrators A to E (Vials 1 to 5) or FALGPA-Solution (Vial 6) provided with the kit.
Sample Preparation
If cell-free aqueous solutions are used as samples, no sample preparation is required. For testing of cell culture samples, the samples must be centrifuged; the supernatants can be used in the test.
In case of samples with an expected Collagenase activity of approximately >1 FALGPA U/ml, or if the rate of photometric absorbance of a sample measurement is higher than the measured rate of absorbance of the highest Calibrator E, then dilute the sample (e.g., 1:20 and 1:100) with 5 mM HEPES, 1 mM CaCl2 pH 7.5, and test the diluted sample.
Test Procedure
The test was developed and evaluated using 5 μl sample or calibrator and 95 μl FALGPA-Solution per well of the microplate. Volumes should not be changed.
Use at least triplicates of each sample, calibrator, and controls. Include the calibrators provided with the kit on the same plate in each run to generate quantitative results. Positive and negative controls should also be measured on the sample plate.
Only thaw the number of 1 ml vials of FALGPA Solution (Vial 6) required for the procedure. For ease of use, the FALGPA Solution can be pooled into a multichannel reagent reservoir and pipetted via 8- or 12- channel pipettes.
  1. Thaw kit contents at +15 to +25°C. As soon as kit contents are thawed, store Calibrators A to E (Vials 1 to 5) on ice and FALGPA Solution (Vial 6) at +15 to +25°C. During the subsequent steps, the solutions must be stored as described here.
  2. Centrifuge all vials for 10 seconds using a tabletop microcentrifuge. Transfer 95 μl of FALGPA Solution (Vial 6) into each well of a microplate that will be used for the reactions, based on the total number of replicates of blank, calibrators, controls, and samples.
  3. Pipette 5 μl per well of each of the following reagents in triplicate to 3 wells each on the microplate in this order:
    - Blank (purified water)
    - Negative control(s) of the sample matrix (cell culture medium or aqueous buffer solution)
    - Positive control(s) of a Liberase Blend
    - Calibrators A to E (Vials 1 to 5)
    - Samples (centrifuged cell culture supernatants or aqueous solutions from your biotechnological process).
  4. Remove air bubbles in the wells by gently tapping the microplate on an even surface. If possible, include a 10 second agitation (shaking) step on the multiwell plate reader on the lowest possible agitation setting to remove any remaining air bubbles before measuring.
  5. Immediately measure the rate of absorbance (e.g., mAbs/min) at 340 nm for 10 minutes.
", "Country": "XG", "Code": "Protocols", "Name": "Protocols" }, { "Language": "en", "Value": "\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n
Vial / BottleCapLabelFunction / DescriptionContent
1Brown            Collagenase Activity Test, Collagenase I
\n\t\t\tCalibrator A              
\n\t\t\t
    \n\t\t\t\t
  • Collagenase I in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for calibration of the assay
  • \n\t\t\t
\n\t\t\t
1 vial,
\n\t\t\t250 μl
2BrownCollagenase Activity Test, Collagenase I
\n\t\t\tCalibrator B
\n\t\t\t
    \n\t\t\t\t
  • Collagenase I in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for calibration of the assay
  • \n\t\t\t
\n\t\t\t
1 vial,
\n\t\t\t250 μl
3BrownCollagenase Activity Test, Collagenase I
\n\t\t\tCalibrator C
\n\t\t\t
    \n\t\t\t\t
  • Collagenase I in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for calibration of the assay
  • \n\t\t\t
\n\t\t\t
1 vial,
\n\t\t\t250 μl
4BrownCollagenase Activity Test, Collagenase I
\n\t\t\tCalibrator D
\n\t\t\t
    \n\t\t\t\t
  • Collagenase I in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for calibration of the assay
  • \n\t\t\t
\n\t\t\t
1 vial,
\n\t\t\t250 μl
5BrownCollagenase Activity Test, Collagenase I
\n\t\t\tCalibrator E
\n\t\t\t
    \n\t\t\t\t
  • Collagenase I in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for calibration of the assay
  • \n\t\t\t
\n\t\t\t
1 vial,
\n\t\t\t250 μl
6YellowCollagenase Activity Test, FALGPA Solution\n\t\t\t
    \n\t\t\t\t
  • 1 mM FALGPA in buffered solution
  • \n\t\t\t\t
  • Ready-to-use solution for colorimetric detection of Collagenase activity
  • \n\t\t\t
\n\t\t\t
10 vials,
\n\t\t\t1 ml each
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "The Collagenase Activity Test kit is based on the chromogenic substrate FALGPA, which is a labeled tetrapeptide resembling the primary structure of collagen, and which is specifically hydrolyzed by all known collagenases but not by other proteases. This test offers a better precision and more convenience compared to other existing tests, and it is perfectly suited for process development and in-process control.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "
    \n\t
  • Aqueous buffer solutions from biotechnological processes.
  • \n\t
  • Cell culture supernatant.\n\t
    If cell culture supernatant is used as sample material, first test a sample of fresh media for potential background reactivity. When testing fresh, unused media, ensure that all supplements required for the culture conditions have been added to the media.
    \n\t
  • \n
", "Country": "XG", "Code": "Sample Materials", "Name": "Sample Materials" }, { "Language": "en", "Value": "
Positive Control
As a positive control, use a freshly prepared solution of any Liberase Blends*. The working concentration should be approximately 0.25 Wünsch Units/ml.
Negative Control
As a negative control, use a Collagenase-free matrix of your sample material, such as cell culture medium or aqueous buffer solution.", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "
  • Reliability
    Accurate and reproducible results due to included calibration standards and high lot-to-lot consistency of the kit.
  • Convenience
    Solutions of FALGPA substrate and Collagenase standards are ready-to-use; no manual preparation of solutions or extraction of dyes needed.
  • High sensitivity
    Limit of detection: 0.32 FALGPA Units/mL.
  • Fast turnaround time
    Time-to-result: 30 minutes.
", "Country": "XG", "Code": "Benefits", "Name": "Benefits" }, { "Language": "en", "Value": "Test kit for determination of collagenase activity for in-process control.", "Country": "XG", "Code": "Positioning", "Name": "Positioning" }, { "Language": "en", "Value": "The kit is designed for determination of collagenase activity in in-process control.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "Measuring range: 0.32 - 1.15 FALGPA Units/mL
Precision: ≤15% (typically <10%)
Specificity: Collagenases I and II from Clostridium histolyticum
Assay time: 30 min approximately
Test format: 96-well plate, absorption at 340 nm
Stability: At -15 to -25°C within specification range for 12 months.", "Country": "XG", "Code": "Specification", "Name": "Specification" } ] } } ] }

Collagenase Activity Test

Kit for determination of the enzymatic activity of Collagenases I and II

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Ordering Information

Technical documents

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