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Collagenase Activity Colorimetric Assay Kit

FOR RESEARCH USE ONLY!       
 
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | tech@biovision.com 
 
Collagenase Activity Colorimetric Assay Kit                                6/14 
(Catalog # K792-100; 100 assays; Store at -20°C) 
I.  Introduction:    
Collagenase (EC 3.4.24.3) is an enzyme in the matrix metalloproteinase family that breaks down collagen, assisting in degradation of the 
extracellular matrix, 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. BioVision’s Collagenase Activity Assay Kit provides a quick and easy way to determine activity of Collagenase. The Kit 
measures collagenase activity using a synthetic peptide (FALGPA) that mimics collagen’s structure. 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. 
II.  Application: 
  Measurement of collagenase activity 
  For screening/studying/characterizing collagenase inhibitors 
III.  Sample Type: 
Purified Collagenase, bacterial extract 
 
 
IV.  Kit Contents: 
 
Components  K792-100  Cap Code  Part Number 
Collagenase Assay Buffer  20 ml  WM  K792-100-1 
Collagenase (0.35 U/ml)  1 ml  Red  K792-100-2 
Collagenase Substrate (FALGPA)  4 ml  NM  K792-100-3 
Inhibitor (1,10-Phenanthroline) [1 M]  50 µl  Yellow  K792-100-4 
 
V.  User Supplied Reagents and Equipment: 
  96-well clear plate with flat bottom  
  Multi-well spectrophotometer 
VI.  Storage Conditions and Reagent Preparation: 
    Store kit at –20°C, protected from light. Briefly centrifuge small vials prior to opening. Read entire protocol before performing the assay.    
  Collagenase Assay Buffer: Bring to room temperature before use. Store at -20°C or 4°C. 
  Collagenase, Collagenase Substrate (FALGPA), and Inhibitor: Ready to use as supplied. Aliquot and store at -20°C. Avoid repeated 
freeze/thaw. Keep on ice during use. Stable for two months. 
VII.  Collagenase Assay Protocol: 
1. Sample  Preparation:  Dissolve  test  collagenase  in  cold  dH2O  or  Hank’s  Balanced  Salt  Solution  (HBSS).  Bacterial  extract  can  be 
prepared  in  cold PBS. Suggested  range  of  collagenase  for measuring  is  0.02-10 mU. Add  2-10  μl  of  test  collagenase  or  bacterial 
extract into desired well(s) in 96-well plate and adjust the volume to 100 μl with Collagenase Assay Buffer. For positive control, add 10 
µl of provided Collagenase (0.35 U/ml). For Inhibitor Control, add 10 µl of provided Collagenase (0.35 U/ml) and 2 µl of Inhibitor (1,10-
Phenanthroline) into desired well(s). Adjust the volume of positive control and inhibitor control wells to 100 μl with Collagenase Assay 
Buffer. For reagent background control, add 100 μl of Collagenase Assay Buffer.     
  Notes: 
a) For unknown collagenase activity, we suggest  testing several amounts of collagenase or bacterial extract  to ensure  the activity  is 
within the assay range.  
b) High activity samples will consume substrate within 3 min. Dilute enzyme and remeasure if necessary.  
2. Inhibitor Screening (optional): To test Collagenase inhibitors, dissolve test inhibitor to 100X in an appropriate solvent. Add 2 µl of test 
inhibitor and 10 µl of provided Collagenase (0.35 U/ml)  into Test  Inhibitor well(s). Prepare a parallel well as Enzyme Control (EC) by 
adding 10 µl of provided Collagenase. Adjust the volume of Test Inhibitor and Enzyme Control wells to 100 μl with Collagenase Assay 
Buffer. Incubate at room temperature for 10 min.  
  Note:  
  Prepare  a  solvent  control well  by  adding  the  same  volume  of  solvent  as  the  test  inhibitor  to  test  its  effect  on Collagenase  activity. 
Provided Inhibitor (1,10-Phenanthroline) does not require a solvent control. 
3. Reaction Mix:  Prepare  enough  Reaction Mix  for  the  number  of  wells  (test  collagenase,  positive  control,  inhibitor  control,  reagent 
background, test inhibitor, and Enzyme Control) to be analyzed. For each reaction, prepare 100 µl reaction mix:  
           
                                   Collagenase Substrate (FALGPA)          40 µl   
      Collagenase Assay Buffer            60 µl 
Mix and add 100 μl of the reaction mix into each well, mix well. 
4. Measurement:  Immediately measure absorbance kinetically at OD 345 nm  in a microplate  reader at 37°C  for 5-15 min. Low activity 
samples can be measured for 1-3 hrs. FOR RESEARCH USE ONLY!       
 
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | tech@biovision.com 
 
0
0.1
0.2
0.3
0.4
enzyme 10 mM (1, 10)-
Phenanthroline
Enzyme activity  
(Units/ml) 
-0.12
-0.1
-0.08
-0.06
-0.04
-0.02
0
0 2 4 6 8 10
OD 345 nm 
time (min) 
0 ul collagenase
2
4
6
8
10
5. Calculation: 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 min. apart.  
To determine Activity, use the following equation: 
 
  (
        
                
                     )  (   )     
(    )     
  U/ml  
 
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 FALGPA 
                            V = Enzyme volume (ml) 
 
        For inhibitor screen, calculate percent inhibition using the following equation: 
           
            (      )         (         )
        (      )
 
                           
         (a)                                                                               (b)                                                             
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
        Figure: (a) Collagenase activity over 10 min. (b) Enzyme activity of collagenase and inhibition by 10 mM (1,10)-Phenanthroline. Assay 
was performed following the kit protocol. 
VIII.    RELATED PRODUCTS: 
  Collagen-I, human recombinant (4796)    MMP-1 Inhibitor Screening Kit (K794)       
  Collagen-III, human recombinant (4797)    MMP-8 (rat) ELISA Kit (K4743)       
  MMP-1 Antibody (5781)        MMP-2 Antibody (5562)       
  MMP-9 Antibody (3529, 3969, 5565)      MMP-9, human recombinant (7789) 
  MMP-11 Antibody (3531R)        MMP-17 Antibody (3537) 
 
                                                                      
      
FOR RESEARCH USE ONLY!  Not to be used on humans. 
 
 
 
 
 
 
 
 
Sample Collagenase Activity 
% Inhibition  X 100 
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