ab Creatine Kinase Activity Assay Kit (Colorimetric)
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1 ab Creatine Kinase Activity Assay Kit (Colorimetric) Instructions for Use For the sensitive and accurate measurement of creatine kinase activity in various samples. This product is for research use only and is not intended for diagnostic use. Version 4 Last Updated 14 April 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 11 ASSAY PROCEDURE and DETECTION 12. ASSAY PROCEDURE and DETECTION 13 DATA ANALYSIS 13. CALCULATIONS TYPICAL DATA 16 RESOURCES 15. QUICK ASSAY PROCEDURE TROUBLESHOOTING FAQ INTERFERENCES NOTES 23 Discover more at 1
3 INTRODUCTION 1. BACKGROUND Creatine Kinase Activity Assay kit (ab155901), creatine kinase converts creatine into phosphocreatine and ADP. The generated phoshocreatine and ADP reacts with CK Enzyme Mix to form an intermediate, which reduces a colorless Probe to a colored product with strong absorbance at 450 nm. This kit is high-throughput adaptable, simple and sensitive. This assay kit can detect Creatine Kinase activity less than 1 mu. Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N-phosphotransferase is a common cellular enzyme. It catalyzes the reversible conversion of creatine and ATP into ADP and phosphocreatine. CK is widely expressed in various tissues and cell types, with highest activity in striated muscles, heart tissue and brain. CK consists of two subunits: M (muscle) and B (brain), and has three isoenzymes: CK-MM (skeleton muscle), CK-MB (cardiac muscle), and CK-BB (brain). Increased CK level is associated with many diseases such as myocardial infarction, muscular dystrophy, pulmonary infarction and brain tumors. Accurate measurement of CK is crucial for early diagnosis, prediction and therapeutic strategy. Creatine + ATP Creatine Kinase Phosphocreatine + ADP CK Enzyme CK ADP Mix Intermediate Developer Color detection (λ = 450nm) Discover more at 2
4 INTRODUCTION 2. ASSAY SUMMARY Standard curve preparation Sample preparation Add reaction mix Measure optical density (OD450 nm) in a kinetic mode (T 1 ) Incubate at 37 C minutes Measure optical density (OD450 nm) in a kinetic mode (T 2 ) 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 -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 4
6 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) CK Assay Buffer 25 ml -20 C -20 C CK Substrate 1 ml -20 C -20 C ATP (Lyophilized) 1 vial -20 C -20 C CK Enzyme Mix (Lyophilized) 1 vial -20 C -20 C CK Developer (Lyophilized) 1 vial -20 C -20 C NADH Standard (Lyophilized) 1 vial -20 C -20 C Positive Control (Lyophilized) 1 vial -20 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 (ddh 2 O) Colorimetric microplate reader equipped with filter for OD450nm 96 well plate:clear plates for colorimetric assay Microcentrifuge Pipettes and pipette tips Heat block or water bath Vortex 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 CK Assay Buffer: Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.2 Substrate: Ready to use as supplied. If necessary, warm in 37 C water bath to dissolve any precipitate, then vortex to mix thoroughly. Aliquot substrate so that you have enough to perform the desired number of assays. Store at -20 C. 9.3 ATP: Reconstitute with 220 µl dh 2 O. Pipette up and down to dissolve completely. Aliquot ATP so that you have enough to perform the desired number of assays. Store at -20 C. Use within 2 months. 9.4 CK Enzyme Mix: Reconstitute with 220 µl CK Assay Buffer. Pipette up and down to dissolve completely. Aliquot enzyme mix so that you have enough to perform the desired number of assays. Store at -20 C. Avoid repeated freeze/thaw cycles. Keep on ice whilst in use. 9.5 CK Developer: Reconstitute with 220 µl dh 2 O. Pipette up and down to dissolve completely. Aliquot developer so that you have enough to perform the desired number of assays. Store at -20 C. Use within 2 months. 9.6 NADH Standard: Reconstitute with 50 µl CK Assay Buffer to generate 10 mm (10.0 nmol/μl) NADH Standard solution. Aliquot standard so that you have enough to perform the desired number of assays. Store at -20 C. Keep on ice while in use. Discover more at 8
10 ASSAY PRE ASSAY PREPARATION 9.7 Positive Control: Reconstitute with 200 µl CK Assay Buffer to generate 10 mu/µl stock and mix thoroughly. Aliquot positive control so that you have enough to perform the desired number of assays. Store at -20 C. Discover more at 9
11 ASSAY PRE 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 Prepare 1mM of NADH Standard by adding 10 µl of 10 mm NADH Standard to 90 µl Assay Buffer Using 1 mm 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 [NADH] 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 50 µl). Discover more at 10
12 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 as well as the deproteinization 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 100 µl ice cold CK Assay Buffer Homogenize cells quickly by pipetting up and down a few times Centrifuge sample for 2 5 minutes at 4 C at top speed 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 = 10 mg) Wash tissue in cold PBS Resuspend tissue in 100 µl of ice cold CK Assay Buffer. Discover more at 11
13 ASSAY PRE ASSAY PREPARATION Homogenize tissue with a Dounce homogenizer sitting on ice, with passes Centrifuge samples for 2 5 minutes at 4 C at top speed using a cold microcentrifuge to remove any insoluble material Collect supernatant and transfer to a clean tube Keep on ice Plasma and Serum Samples: Plasma and serum 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), since it depends on the amount of active CK enzyme in the sample. NOTE: We suggest using different volumes of sample to ensure readings are within the Standard Curve range. NOTE: Small molecules such as ADP, NADH etc. in some tissue samples such as liver may generate background. To remove small molecules, we suggest using 10 KDa spin column. Discover more at 12
14 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 Set up Reaction wells: - Standard wells = 50 µl standard dilutions. - Sample wells = 1 50 µl samples (adjust volume to 50 µl/well with Assay Buffer). - Positive Control = 2-10 µl Positive Control (adjust volume to 50 µl/well with Assay Buffer). - Background wells = 50 µl Assay Buffer Reaction Mix: Prepare Reaction Mix for each reaction Component Colorimetric Reaction Mix (µl) CK Assay Buffer 34 CK Enzyme Mix 2 CK Developer 2 ATP 2 CK Substrate 10 Mix enough reagents for the number of assays (samples, standards, positive control and background 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 + positive control +1) Add 50 µl of Reaction Mix to each well. Mix well Measure output (A 1 ) at T 1 on a microplate reader. - Colorimetric assay: measure OD450 nm Discover more at 13
15 ASSAY PRE ASSAY PROCEDURE and DETECTION 12.5 Incubate at 37 ºC for minutes* Measure output (A 2 ) at T 2 on a microplate reader. - Colorimetric assay: measure OD450 nm. *NOTE: Incubation time depends on the Creatine Kinase activity in the samples. We recommend measuring the OD (A 1 & A 2 ) in a kinetic mode and choose two time points (T 1 & T 2 ) in the linear range to calculate the CK activity of the samples. The NADH standard curve can read in endpoint mode (i.e. at the end of incubation time). Discover more at 14
16 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 Subtract the mean absorbance value of the blank (Standard #1) from all standard and sample readings. This is the corrected absorbance 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) Calculate the Creatine Kinase activity of the test sample. ΔOD = A 2 A Extrapolate sample readings from the standard curve plotted using the following equation: B = ( ΔOD (y intercept) Slope ) 13.6 CK activity (as nmol/min/ml or mu/ml) in the test samples is calculated as: Creatine Kinase Activity = ( B ΔT x V) Dilution Factor Where: B = NADH amount from the standard curve (nmol). ΔT = reaction time (min). V = sample volume added into the reaction well (ml). Discover more at 15
17 DATA ANALYSIS Unit Definition: One unit of Creatine Kinase is the amount of enzyme that will generate 1.0 μmol of NADH per min at ph 9.0 at 37 C. 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 NADH standard calibration curve using colorimetric reading. Discover more at 16
18 DATA ANALYSIS Figure 2: Creatine kinase activity tested in 5 µl Human serum and 192 ng rat heart lysate. Discover more at 17
19 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 standard, ATP, developer, positive control and prepare enzyme mix (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 (50 µl), samples (50 µl), and positive control (50 µl). Prepare Reaction Mix (Number samples + standards + positive control + 1). Component Add 50 µl Reaction Mix to each reaction well. Measure plate (A 1 ) at T 1, at OD450 nm. Incubate plate 37 ºC mins. Measure plate (A 2 ) at T 2, at OD450 nm. Colorimetric Reaction Mix (µl) CK Assay Buffer 34 CK Enzyme Mix 2 CK Developer 2 ATP 2 CK Substrate 10 Discover more at 18
20 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 Colorimeters: Clear plates Fluorometric: black wells/clear bottom plate Use PCA precipitation protocol for deproteinization Use Dounce homogenizer (increase number of strokes); observe for lysis under microscope 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 19
21 RESOURCES Problem Cause Solution Pipetting errors in standard or reaction mix Standard readings do not follow a linear pattern Unanticipated results 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 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 as to be in the linear range Discover more at 20
22 RESOURCES 17.FAQ What is the sensitivity of this assay? The assay has a detection sensitivity of 0.5 mu/ml of glutathione peroxidase. Which protein assay is compatible with this kit? We suggest you use a detergent compatible BCA assay kit: Is using fresh samples mandatory? We always recommend to use fresh samples over frozen for the best results. However if needed, tissue can be flash-frozen in liquid nitrogen and then stored at -80 ºC or cells/serum/plasma can be stored at -80 ºC and used with this kit. We recommend minimizing freeze-thaw cycles for the best results. What is the sample volume to be used with this kit for plasma samples from rat? This depends on the amount of active CK enzyme in the sample. The sample volume per well would need to be optimized to make sure that the values obtained are within the linear range of the standard curve. Discover more at 21
23 RESOURCES 18.INTERFERENCES These chemicals or biological materials will cause interferences in this assay causing compromised results or complete failure Small molecules e.g. ADP, NADH etc in some tissue samples e.g. liver may generate background. Discover more at 22
24 RESOURCES 19.NOTES Discover more at 23
25 RESOURCES Discover more at 24
26 RESOURCES Discover more at 25
27 RESOURCES Discover more at 26
28 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2015 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. RESOURCES All information / detail is correct at time of going to print. 27
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