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 Discover more at www.abcam.com 14 16 18 19 20 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. Discover more at www.abcam.com 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. Discover more at www.abcam.com 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. Discover more at www.abcam.com 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 Discover more at www.abcam.com 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. Discover more at www.abcam.com 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 Discover more at www.abcam.com 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. Discover more at www.abcam.com 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. Discover more at www.abcam.com 9 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 Discover more at www.abcam.com 10 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. Discover more at www.abcam.com 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) Discover more at www.abcam.com 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. Discover more at www.abcam.com 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 Discover more at www.abcam.com 14 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. Discover more at www.abcam.com 15 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 Discover more at www.abcam.com 16 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 Discover more at www.abcam.com Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range 17 RESOURCES 16.FAQ Discover more at www.abcam.com 18 RESOURCES 17.INTERFERENCES Discover more at www.abcam.com 19 RESOURCES 18.NOTES Discover more at www.abcam.com 20 RESOURCES Discover more at www.abcam.com 21 RESOURCES Discover more at www.abcam.com 22 UK, EU and ROW Email: technical@abcam.com | Tel: +44-(0)1223-696000 Austria Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259 France Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96 Germany Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154 Spain Email: soportecientifico@abcam.com | Tel: 911-146-554 Switzerland Email: technical@abcam.com Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530 US and Latin America Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226) Canada Email: ca.technical@abcam.com | Tel: 877-749-8807 China and Asia Pacific Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通) Japan Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940 www.abcam.com | www.abcam.cn | www.abcam.co.jp Copyright © 2015 Abcam, All Rights Reserved. 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