ab83464 Catalase Assay Kit (Colorimetric/Fluorometric)

Transcription

1 ab83464 Catalase Assay Kit (Colorimetric/Fluorometric) Instructions for Use For the rapid, sensitive and accurate measurement of catalase activity in various samples. This product is for research use only and is not intended for diagnostic use. Version 7 Last Updated 7 May 2015

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION 12 ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION 14 DATA ANALYSIS 13. CALCULATIONS TYPICAL DATA 17 RESOURCES 15. QUICK ASSAY PROCEDURE TROUBLESHOOTING FAQ INTERFERENCES NOTES 27 Discover more at 1

3 INTRODUCTION 1. BACKGROUND Catalase Assay Kit (colorimetric/fluorometric) (ab83464) provides a highly sensitive, simple, direct and HTS ready assay for measuring catalase activity in biological samples. In the assay, catalase first reacts with H 2 O 2 to produce water and oxygen, the unconverted H 2 O 2 reacts with OxiRed probe to produce a product, which can be measured at 570 nm (Colorimetric method) or at Ex/Em = 535/587 nm (fluorometric method). Catalase activity is reversely proportional to the signal. The kit can detect 1 µu or less of catalase activity in samples. Catalase is a ubiquitous antioxidant enzyme that is present in nearly all living organisms. It functions to catalyze the decomposition of hydrogen peroxide (H 2 O 2 ) to water and oxygen. Discover more at 2

4 INTRODUCTION 2. ASSAY SUMMARY Standard curve preparation and add stop solution Sample preparation, add H 2 O 2 and incubate 25 C 30 mins Add stop solution to samples Add development mix and incubate 25 C for 10 min Measure optical density (OD570 nm) or fluorescence (Ex/Em = 535/587 nm) Discover more at 3

5 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 4ºC 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 4

6 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) Catalase Assay Buffer 25 ml 4 C 4 C OxiRed probe (in DMSO) 200 µl 4 C 4 C HRP (lyophilized) 1 vial 4 C 4 C H 2 O 2 (0.88 M) 25 µl 4 C 4 C Stop Solution 1 ml 4 C 4 C Catalase Positive Control 2 µl 4 C 4 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 (ddh 2 O) PBS Microcentrifuge Pipettes and pipette tips Colorimetric or fluorescent microplate reader equipped with filter for OD 570 nm or Ex/Em = 535/587 nm (respectively) 96 well plate: clear plates for colorimetric assay; black plates (clear bottoms) for fluorometric assay Heat block or water bath Dounce homogenizer or pestle (if using tissue) Discover more at 5

7 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 6

8 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, heat labile components and samples on ice during the assay. Make sure all buffers and solutions are at room temperature before starting the experiment. Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. 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 right type of plate for your detection method of choice. Make sure the heat block/water bath and microplate reader are switched on. Discover more at 7

9 ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening. 9.1 Catalase Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at 4 C. 9.2 OxiRed Probe in DMSO: Ready to use as supplied. Warm by placing in a 37 C bath for 1 5 minutes to thaw the DMSO solution before use. NOTE: DMSO tends to be solid when stored at -20 C, even when left at room temperature, so it needs to melt for few minutes at 37 C. Aliquot probe so that you have enough volume to perform the desired number of assays. Store at 4 C protected from light and moisture. Once the probe is thawed, use with two months. Keep on ice while in use. 9.3 HRP: Reconstitute in 220 µl Assay Buffer. Aliquot HRP so that you have enough volume to perform the desired number of assays. Store at 4 C protected from light. Use within two months. Keep on ice while in use. 9.4 H 2 O 2 Standard: Ready to use as supplied. Aliquot standard so that you have enough volume to perform the desired number of assays. Store at 4 C protected from light. Use within two months. Keep on ice while in use. 9.5 Stop Solution: Ready to use as supplied. Aliquot stop solution so that you have enough volume to perform the desired number of assays. Store at 4 C protected from light. Use within two months. Keep on ice while in use. Discover more at 8

10 ASSAY PRE ASSAY PREPARATION 9.6 Catalase Positive Control: Dilute the Catalase Positive Control with 500 µl of Assay Buffer. Aliquot positive control so that you have enough volume to perform the desired number of assays. Store at -20 C for 2 months or 2-3 days at 4 C. Keep on ice while in use. Discover more at 9

11 ASSAY PREPARATION 10.STANDARD PREPARATION Always prepare a fresh set of standards for every use. Diluted standard solution is unstable and must be used within 4 hours For the colorimetric assay: Prepare a 20 mm H 2 O 2 standard by diluting 5 µl of the provided 0.88 M H 2 O 2 standard with 215 µl of ddh 2 O Prepare a 1 mm H 2 O 2 standard by diluting 50 µl of the 20 mm H 2 O 2 standard with 950 µl of ddh 2 O Using 1 mm H 2 O 2 standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End [Catalase] in well nmol/well nmol/well nmol/well nmol/well nmol/well nmol/well Each dilution has enough amount of standard to set up duplicate readings (2 x 90 µl) Add 10 µl of Stop Solution into each standard well. Discover more at 10

12 ASSAY PRE ASSAY PREPARATION 10.2 For the flurometric assay: Prepare a 20 mm H 2 O 2 standard by diluting 5 µl of the provided 0.88 M H 2 O 2 standard with 215 µl of ddh 2 O Prepare a 1 mm H 2 O 2 standard by diluting 5 µl of the 20 mm H 2 O 2 standard with 95 µl of ddh 2 O Prepare a 0.1 mm H 2 O 2 standard by diluting 10 µl of the 1 mm H 2 O 2 standard with 90 µl of ddh 2 O Using 0.1 mm H 2 O 2 standard, prepare standard curve dilution as described in the table in a microplate or microcentrifuge tubes: Standard # Volume of Standard (µl) Assay Buffer (µl) Final volume standard in well (µl) End [Catalase] in well nmol/well nmol/well nmol/well nmol/well nmol/well nmol/well Each dilution has enough amount of standard to set up duplicate readings (2 x 90 µl) Dilute provided Stop Solution 1:10 with Assay Buffer e.g.10 µl Stop Solution in 90 µl Assay Buffer Add 10 µl of diluted Stop Solution into each standard well. NOTE: If your sample readings fall out the range of your fluorometric standard curve, you might need to adjust the dilutions and create a new standard curve. Discover more at 11

13 ASSAY PRE ASSAY PREPARATION 11.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 Cell (adherent or suspension) samples: Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 10 6 cells) Wash cells with cold PBS Resuspend cells in 200 µl of ice cold Assay Buffer Homogenize cells quickly by pipetting up and down a few times Centrifuge sample for 15 minutes at 4 C at 10,000 x g using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Tissue samples: Harvest the amount of tissue necessary for each assay (initial recommendation = 100 mg) Wash tissue in cold PBS Resuspend tissue in 100 µl of ice cold Assay Buffer. Discover more at 12

14 ASSAY PRE ASSAY PREPARATION Homogenize tissue with a Dounce homogenizer sitting on ice, with passes Centrifuge samples for 15 minutes at 4 C at 10,000 x g using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Plasma, Serum and Urine and other biological fluids: Serum and urine samples can be tested directly by adding sample to the microplate wells. However, to find the optimal values and ensure your readings will fall within the standard values, we recommend performing several dilutions of the sample (1/2 1/5 1/10) Erythrocytes: Harvest the amount of cells necessary for each assay (initial recommendation = 200 µl) Wash cells with cold PBS Resuspend cells in 200 µl of ice cold Assay Buffer Homogenize cells quickly by pipetting up and down a few times Centrifuge sample for 15 minutes at 4 C at 10,000 x g using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice. NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. Discover more at 13

15 ASSAY PROCEDURE and DETECTION 12.ASSAY PROCEDURE and DETECTION Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate. For unknown samples, we recommend testing several doses of your sample to ensure the readings are within the linear range Set up Reaction wells: - Standard wells = 90 µl standard dilutions. - Sample wells = 2 78 µl samples (adjust volume to 78 µl/well with Assay Buffer). - Sample High Control (HC) = 2 78 µl samples (adjust volume to 78 µl/well with Assay Buffer). - Positive control = 1 5 µl Positive control (adjust volume to 78 µl/well with Assay Buffer Sample High Controls: - Add 10 µl stop solution to one well of each sample pair. - Mix. Incubate 25 C for 5 minutes to completely inhibit the catalase in the sample Catalase Reaction: - For colorimetric assay: Add 12 µl fresh 1 mm H 2 O 2 into each well of both samples and sample HC to start the reaction. - For fluorometric assay: Add 1.5 µl fresh 1 mm H 2 O 2 into each well of both samples and sample HC and 13.5 µl Assay Buffer, to start the reaction. - Incubate at 25 C for 30 minutes. - Add 10 µl Stop Solution into each sample and positive control wells to stop the reaction. Discover more at 14

16 ASSAY PRE ASSAY PROCEDURE and DETECTION NOTE: High control and Standard curve wells already contain Stop Solution Development Mix: Prepare 50 µl of Development Mix for each reaction Component Colorimetric Reaction Mix (µl) NOTE: *For fluorometric readings, using 0.3 μl/well of the probe decreases the background readings, therefore increasing detection sensitivity. Mix enough reagents for the number of assays (samples, standard and positive 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 + Standards +1) Add 50 µl of Development Mix into each sample, sample high control, standard and positive control wells Mix and incubate at 25 C for 10 minutes protected from light Measure output on a microplate reader. - Colorimetric assay: measure OD570 nm. Fluorometric Reaction Mix (µl) Assay Buffer OxiRed Probe 2 0.3* HRP Solution Fluorometric assay: measure Ex/Em = 535/587 nm. Discover more at 15

17 DATA ANALYSIS 13.CALCULATIONS Samples producing signals greater than that of the highest standard should be further diluted in appropriate buffer and reanalyzed, then multiplying the concentration found by the appropriate dilution factor. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates) Average the duplicate reading for each standard and sample Signal changes by catalase in samples is: A = A HC A sample Use ΔA to the H 2 O 2 standard curve to get B nmol of H 2 O 2 decomposed by catalase in 30 minutes reaction Plot the corrected absorbance values for each standard as a function of the final concentration of catalase Draw the best smooth curve through these points to construct the standard curve. Most plate reader software or Excel can plot these values and curve fit. Calculate the trendline equation based on your standard curve data (use the equation that provides the most accurate fit) Catalase activity (in nmol/min/ml or mu/ml) in the test samples is calculated as: Catalase Activity = ( B 30 x V) D Where: B = Decomposed H 2 O 2 amount (nmol) for H 2 O 2 standard curve. V = Pretreated sample volume (ml) added into the reaction well. 30 = Reaction time is 30 minutes. Unit definition: One unit of catalase is the amount of catalase decomposes 1.0 µmol of H 2 O 2 per minute at ph 4.5 at 25 C. Discover more at 16

18 DATA ANALYSIS Discover more at 17

19 DATA ANALYSIS 14.TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Figure 1. Typical H 2 O 2 standard calibration curve using colorimetric reading. Discover more at 18

20 DATA ANALYSIS Figure 2: Sample tested using catalase assay kit (ab83464) in colorimetric assay. Discover more at 19

21 RESOURCES 15.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 buffer, probe, HRP solution, H 2 O 2, and positive control (aliquot if necessary); get equipment ready. Prepare appropriate standard curve for your detection method of choice (colorimetric or fluorometric). Prepare samples in duplicate (find optimal dilutions to fit standard curve readings). Set up plate for standard (78 µl), samples (78 µl), positive control and sample high control (HC) (78 µl). To Sample High Controls and standards only, add 10 µl stop solution. Mix and incubate 25 C for 5 mins. For colorimetric assay: add 12 µl 1 mm H 2 O 2 into each well of both samples and sample HCs. For fluorometric assay: add 1.5 µl 1 mm H 2 O 2 into each well of both samples and sample HCs and 13.5 µl Assay Buffer. Incubate 25 C 30 mins. Add 10 µl stop solution into each sample and positive control wells. Prepare Development Mix (Number samples + standards + sample HC + 1). Component Colorimetric Reaction Mix (µl) Fluorometric Reaction Mix (µl) Assay Buffer OxiRed Probe HRP Solution 2 2 Add 50 µl Development Mix to the sample, standard, sample HC and positive control wells. Incubate plate 25 C 10 mins protected from light. Discover more at 20

22 RESOURCES Measure plate at OD 570 nm for colorimetric assay or Ex/Em= 535/587 nm for fluorometric assay. Discover more at 21

23 RESOURCES 16.TROUBLESHOOTING Problem Cause Solution Assay not working Sample with erratic readings Lower/ Higher readings in samples and Standards Use of ice-cold buffer Plate read at incorrect wavelength Use of a different 96- well 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 Improperly thawed components Allowing reagents to sit for extended times on ice Incorrect incubation times or temperatures Buffers must be at room temperature Check the wavelength and filter settings of instrument Colorimetric: Clear plates Fluorometric: black wells/clear bottom plate 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 Thaw all components completely and mix gently before use Always thaw and prepare fresh reaction mix before use Verify correct incubation times and temperatures in protocol Discover more at 22

24 RESOURCES Problem Cause Solution Standard readings do not follow a linear pattern Unanticipated results Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Measured at incorrect wavelength Samples contain interfering substances Sample readings above/ below the linear range Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Always refer to dilutions on protocol Check equipment and filter setting Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range Discover more at 23

25 RESOURCES 17.FAQ What is the sensitivity of this assay and how can plasma/whole blood samples be processed for this assay? The lower end of sensitivity is high pico-units to 1 micro-unit of Catalase activity. What is the activity level of the positive control? How can we increase its value to be comparable with our samples? The positive control is only a benchmark sample. As long as the values are within the range of the standard curve this is fine. The positive control is not be used to compare values with the samples. The positive control is provided to validate that the assay components are all working. The customer can add more volume to get higher values but this is not necessary as long as the values are within the std. curve range. Catalase is a very vulnerable enzyme to freeze-thaw and can lose activity with storage over time. Is sonication enough to lyse cells? We recommend homogenizing cells. This method is gentle yet effective. If needed sonication can be used in addition to homogenization. Generation of heat during sonication affects the activity of the Catalase enzyme. Can samples from bacteria work with this kit? Our kits are developed with mammalian samples but they work with a variety of samples from different species including bacteria. For gram positive bacteria, lysis reagents (Lysozyme treatment) might be required to rupture the cell wall. Gram negative bacterial cells can simply be homogenized as described in the protocol. We recommend testing different volumes/dilutions of the samples to make sure the final readings are within the linear range of the standard curve. Discover more at 24

26 RESOURCES Can food samples be used? Food samples can be homogenized with the assay buffer and then centrifuged to collect the supernatant which will be the sample for the assay. Liquid food samples can be tested without any preparation. All samples must be spun down to make sure there is no floating debris or particulate material. We suggest testing several volumes of the samples to optimize the amount needed to get numbers in the linear range of the std. curve. Can this assay be run in cuvettes? All our assays are optimized based on 96-well plate format. If you wish to run the assays in cuvettes, fewer numbers of samples can be assayed since the volume of the cuvette will need scaling up for all components. The RFU values are same for increasing volumes of our sample. Why? The classic cue to saturation is that when you add more sample the values decrease, meaning the maximum has already been attained and there is limitation of either reagents or Vmax has been reached already. When there is very high amount of catalase in the sample, all the substrate is quickly converted into product and then substrate is no longer available limiting the color development. When you dilute the sample, there is less catalase and hence the substrate is gradually converted to product showing a gradual increase over time. Sample volume needs to be optimized to make sure that just enough is used to get values in the linear range of the std. curve, not too high or too low. Discover more at 25

27 RESOURCES 18.INTERFERENCES These chemicals or biological materials will cause interferences in this assay causing compromised results or complete failure: RIPA: contains SDS which can destroy/decrease the activity of the enzyme. β-mercaptoethanol. (keep below 5 µm). DDT (keep below 5 µm). Other reducing agents in samples. Discover more at 26

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