ab Complex I Enzyme Activity Microplate Assay Kit (Colorimetric)

ab109721 Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) Instructions for use: For the analysis of Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts. This product is for research use only and is not intended for diagnostic use. Version 9b Last Updated 10 August 2017

Table of Contents INTRODUCTION 1 1. BACKGROUND 1 2. ASSAY SUMMARY 2 GENERAL INFORMATION 3 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 3 5. LIMITATIONS 4 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIRED, NOT SUPPLIED 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 7 9. REAGENT PREPARATION 7 10. SAMPLE PREPARATION 8 ASSAY PROCEDURE 11 11. ASSAY PROCEDURE 11 DATA ANALYSIS 13 12. CALCULATIONS 13 13. TYPICAL DATA 15 RESOURCES 17 14. QUICK ASSAY PROCEDURE 17 15. TROUBLESHOOTING 18 16. INTERFERENCES 20 17. FAQS 20 18. APPENDIX MITOCHONDRIAL PURIFICATION PROTOCOL 22 19. NOTES 24

INTRODUCTION INTRODUCTION 1. BACKGROUND Abcam s Complex I Enzyme Activity Microplate Assay Kit (ab109721) is designed for the analysis of mitochondrial OXPHOS Complex I (NADH dehydrogenase) enzyme activity from human, rat, mouse and bovine cell and tissue extracts. This kit recognizes Complex I in human, rat, mouse and bovine cell and tissue extracts. Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the pre-coated microplate wells. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of the provided dye ( = 25.9/mM/well) which leads to increased absorbance at OD 450 nm. An accurate measurement of the enzyme s functional state is achieved by analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables. Please note that this activity assay measures the NADH-dependent activity of Complex I. This activity is not dependent on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay. However assembly deficiencies of Complex I can affect this activity assay. OXPHOS Complex I (NADH dehydrogenase, E.C. 1.6.5.3) activity is controlled by enzyme amount and by post-translational phosphorylation at key specific regulatory residues. Cellular metabolism governs these two factors. Ultimately, the cell type and growth conditions will affect Complex I activity measurements. ab109721 Complex I Enzyme Activity Assay Kit 1

INTRODUCTION 2. ASSAY SUMMARY Sample preparation (5.5 mg/ml) Load sample(s) on plate Incubate for 3 hours at RT Add 200 µl of Assay Solution to each well Measure Optical Density (OD450 nm) in a kinetic mode at RT for 30 minutes* *For kinetic mode detection, incubation time given in this summary is for guidance only. ab109721 Complex I Enzyme Activity Assay Kit 2

GENERAL INFORMATION 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. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handle with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipet by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 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 the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. ab109721 Complex I Enzyme Activity Assay Kit 3

GENERAL INFORMATION 5. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. 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. 6. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) Storage Condition (After Preparation) 20X Wash Buffer 25 ml 4 C 4 C 10X Blocking Buffer 10 ml 4 C 4 C 10X Detergent 1 ml 4 C 4 C 20X NADH (40 mm; 1 vial 4 C -80 C lyophilized) 100X Dye (lyophilized) 1 vial 4 C -80 C 96-well microplate (12 x 8 well strips) 1 4 C 4 C ab109721 Complex I Enzyme Activity Assay Kit 4

GENERAL INFORMATION 7. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring absorbance at OD 450 nm. MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions Tubes for the preparation of reagents and buffer solutions Dounce homogenizer (if using tissue) Method for determining protein concentration: we recommend BCA Protein Quantification Kit (ab102536) For mitochondria isolation: Mitochondria Isolation Kit for Cultured Cells (ab110170) Mitochondria Isolation Kit for Tissue (ab110168) or Mitochondria Isolation Kit for Tissue (with Dounce Homogenizer) (ab110169) ab109721 Complex I Enzyme Activity Assay Kit 5

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. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. 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 all reagents and solutions are at the appropriate temperature before starting the assay. Samples which generate values that are greater than the most concentrated standard should be further diluted in the appropriate sample dilution buffer. Ensure plates are properly sealed or covered during incubation steps. Make sure you have the right type of plate for your detection method of choice. Make sure all necessary equipment is switched on and set at the appropriate temperature. ab109721 Complex I Enzyme Activity Assay Kit 6

ASSAY PREPARATION ASSAY PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening 9.1. 20X Wash Buffer: Prepare 1X Buffer by diluting 20X Buffer in ddh 2 O: to make 500 ml 1X Buffer, combine 25 ml 20X Buffer with 475 ml ddh 2 O. Mix thoroughly and gently. Label this mixture as Buffer. Store Buffer at 4 C. Equilibrate to room temperature before use. 9.2. 10X Blocking Solution: Dilute 10X Blocking Solution in Buffer to create Incubation Solution: to make 100 ml Incubation Buffer, combine 10 ml of 10X Blocking Solution with 90 ml of Buffer. Mix thoroughly and gently. Label this mixture as Incubation Solution. Store Incubation Solution at 4 C. Equilibrate to room temperature before use. 9.3. 10X Detergent: Ready to use as supplied. Equilibrate to room temperature before use. Store at 4 C. 9.4. 20X NADH: Reconstitute the lyophilized NADH in 1.1 ml of ddh 2 O and vortex thoroughly until dissolved. Aliquot reconstituted NADH so that you have enough volume to perform the desired number of assays. Store at -80 C. Reconstituted NADH is stable for up to 6 months. Keep on ice while in use. 9.5. 100X Dye: Reconstitute the dye in 250 µl of ddh 2 O and vortex thoroughly until dissolved. Aliquot reconstituted Dye so that you have enough volume to perform the desired number of assays. Reconstituted Dye is stable for up to 6 months. Store at -80 C. Keep on ice while in use. 9.6. 96-well microplate (12 x 8-well strips): ab109721 Complex I Enzyme Activity Assay Kit 7

ASSAY PREPARATION Ready to use as supplied. This plate can be broken into 12 separate 8-well strips for convenience. Equilibrate to room temperature before use. Store at 4 C. ab109721 Complex I Enzyme Activity Assay Kit 8

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 samples 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. Treat cells with Complex I activators / inhibitors as per your experimental requirements. 10.1. Preparation of extracts from cells (adherent or suspension): 10.1.1. Harvest suspension cells by centrifugation or scrape to collect adherent cells from a confluent culture flask (initial recommendation = 1 2 x 10 7 cells). 10.1.2. Wash cells twice with PBS. 10.1.3. Resuspend and dilute the cell pellet with 9 volumes of PBS (e.g. 50 µl pellet + 450 µl PBS to a total volume of 500 µl). 10.1.4. Optional: Determine the sample protein concentration (using a standard methods such as BCA), by extracting a portion of your sample. Adjust concentration of the sample with PBS so that the final sample protein concentration is 5.5 mg/ml. 10.1.5. Extract the proteins from the sample by adding 10X Detergent solution to sample to a final dilution of 1/10 (e.g. if the total sample volume is 500 µl add 50 µl of 10X Detergent solution). Mix well. 10.1.6. Incubate the tube on ice for 30 minutes to allow solubilization. ab109721 Complex I Enzyme Activity Assay Kit 9

ASSAY PREPARATION 10.1.7. Centrifuge the sample for 20 minutes at 4 C at 12,000 16,000 x g in a cold centrifuge. 10.1.8. Collect supernatant and transfer to a clean tube. Please note the sample concentration now is 5 mg/ml. 10.1.9. Dilute your samples to the desired concentration in Incubation Solution (from step 9.2). Table 1 indicates a typical linear range for the assay. 10.2. Preparation of extracts from tissue: 10.2.1. Harvest tissue for the assay (initial recommendation = 100 200 mg). 10.2.2. Wash tissue thoroughly in cold PBS to remove blood. 10.2.3. Resuspend tissue in 500 µl 1 ml of ice cold PBS. 10.2.4. Homogenize tissue with a Dounce homogenizer sitting on ice, with 20 40 passes, or until sample is fully homogenized and is completely smooth. 10.2.5. Collect homogenate and transfer to a clean tube. 10.2.6. Determine the sample protein concentration (using a standard methods such as BCA) by extracting a portion of your sample. Adjust concentration of the sample with PBS so that the final sample protein concentration is 5.5 mg/ml. 10.2.7. Extract the proteins from the sample by adding 10X Detergent solution to sample to a final dilution of 1/10 (e.g. if the total sample volume is 500 µl add 50 µl of 10X Detergent solution). Mix well. 10.2.8. Incubate the tube on ice for 30 minutes to allow solubilization. 10.2.9. Centrifuge the sample for 20 minutes at 4 C at 12,000 16,000 x g in a cold centrifuge. 10.2.10. Collect supernatant and transfer to a clean tube. Please note the sample concentration now is 5 mg/ml. ab109721 Complex I Enzyme Activity Assay Kit 10

ASSAY PREPARATION 10.2.11. Dilute your samples to the desired concentration in Incubation Solution (from step 9.2). Table 1 indicates a typical linear range for the assay. Sample Type Recommended Concentration (µg/200 µl volume) Cell culture extracts 25 250 Fibroblast (MRC5) cell extracts 100 Heptoblastoma (HepG2) cell extracts 200 Tissue extracts (mitochondria) 5 100 Heart extracts 20 Liver extracts 50 Table 1. Typical ranges of measurement per 200 µl well volume. 10.3. Preparation of isolated mitochondria: You can isolate mitochondria using mitochondrial isolation kits such Mitochondria Isolation Kit for Cultured Cells (ab110170) or Mitochondria Isolation Kit for Tissue (with Dounce Homogenizer) (ab110169). Alternatively, mitochondria can be prepared by simple differential centrifugation of homogenized tissue samples please see Section 17 for a general mitochondrial purification protocol. ab109721 Complex I Enzyme Activity Assay Kit 11

ASSAY PROCEDURE ASSAY PROCEDURE 11.ASSAY PROCEDURE Equilibrate all materials and prepared reagents to correct temperature prior to use. We recommend that you include a positive control sample (untreated samples or sample of known robust complex I activity see FAQ section) in addition to a buffer only control and to assay all samples/controls in duplicate. Prepare all reagents, working standards, and samples as directed in the previous sections. 11.1. Plate Loading: - Sample wells = Add 200 µl of sample prepared as described in the Sample Preparation section to each well of the microplate that will be used for this experiment. - Background/Buffer only control wells = Add 200 µl 1X Incubation buffer to buffer only wells. - Positive control sample wells = Add 200 µl of positive sample to appropriate wells. 11.1.1. Incubate microplate for 3 hours at room temperature. 11.2. Measurement: 11.2.1. Empty the wells by turning the plate over and shaking out any remaining liquid. Blot the plate face down on paper towel. 11.2.2. Add 300 µl of 1X Buffer solution to each well used. 11.2.3. Empty the wells of the microplate by turning the plate over and shaking out any remaining liquid. Blot the plate face down on paper towel. 11.2.4. Add 300 µl of 1X Buffer solution to each well used. 11.3. Prepare Assay Solution: 11.3.1. Prepare 1.75 ml/strip of Assay Solution according to the following table: ab109721 Complex I Enzyme Activity Assay Kit 12

ASSAY PROCEDURE Number of 1X Buffer 20X NADH 100X strips (ml) (µl) Dye (µl) 1 1.67 84 17 2 3.33 167 33 3 5 250 50 4 6.67 333 67 5 8.33 417 83 6 10 500 100 7 11.67 583 117 8 13.33 667 133 9 15 750 150 10 16.67 833 167 11 18.33 917 183 12 20 1,000 200 11.3.2. Empty the wells of 1X Buffer as described in step 11.2.3. 11.3.3. Add 200 µl of Assay Solution (from step 11.3.1) to each well carefully to avoid bubbles. Any bubbles should be popped with a fine needle as rapidly as possible. 11.3.4. Place the plate in the reader and record with the following kinetic program. Mode Wavelength: Time: Interval: Shaking: Temperature Kinetic 450 nm 30 minutes 20 sec - 1 min Shake between readings Room temperature NOTE: Sample incubation time can vary depending on enzyme activity in the samples. 11.3.5. Save data and analyze as described in the Data Analysis section. ab109721 Complex I Enzyme Activity Assay Kit 13

DATA ANALYSIS DATA ANALYSIS 12.CALCULATIONS Extinction coefficient for dye () = 25.9/mM/well Complex I activity in each well is proportional to the increase in absorbance at OD 450 nm within each well. The activity is expressed as the change in absorbance per minute per amount of sample loaded into the well. If the sample background control is significant, then subtract the sample background control from the sample reading. Examine the linear rate of increase in absorbance at OD 450 nm over time. An example is shown below where the rate/slope is calculated between these time points. Most microplate software of performing this function. Repeat this for all samples. Figure 1: Raw data: change in OD 450 nm observed in cultured HepG2 cell lysate. ab109721 Complex I Enzyme Activity Assay Kit 14

DATA ANALYSIS Raw data (as seen in Figure 1) can be expressed as rate (mod/min) per µg of cell lysate added per well as shown below in Figure 2. Figure 2: Complex I activity measured in cultured HepG2 cell lysate. TYPICAL SAMPLE VALUES PRECISION Intra Assay Inter Assay n= 60 60 CV (%) <10 <15 ab109721 Complex I Enzyme Activity Assay Kit 15

DATA ANALYSIS 13. TYPICAL DATA Data provided for demonstration purposes only. Figure 3: Complex I activity measured in normal and Rho0 human fibroblasts. Rho0 cells are cells in which the mitochondrial DNA has been removed and therefore essential Complex I proteins are not expressed. As shown in the right column, the rho0 cells showed no/little complex I activity. Rat cardiomyocytes treated with 40 μm chloramphenicol (CAM) for 5 days. Figure 4: Complex I activity measured in rat cardiomyocytes grown for 5 days in absence or presence of 40 µm chloramphenicol (CAM) to inhibit mitochondrial protein synthesis. In this case, both Complex I assembly and activity was greatly reduced. ab109721 Complex I Enzyme Activity Assay Kit 16

DATA ANALYSIS Figure 5: ab109721 measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples within the recommended ranges given in the protocol. (Note that these ranges depend on mitochondria preparation quality.) Examples of Complex I activity measured in different rat and mouse mitochondrial samples are shown in the graphs above. ab109721 Complex I Enzyme Activity Assay Kit 17

RESOURCES 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. 14.1. Prepare Sample (approximately 1 hour) Adjust sample concentration to 5.5 mg/ml in PBS. Perform Detergent extraction with 1/10 volume 10X Detergent. Incubate on ice for 30 minutes. Centrifuge at 12,000 x g for 20 minutes at 4 C and then collect supernatant. 14.2. Load Plate (3 hours) Load sample(s) on plate being sure to include a normal sample and a buffer control as null reference. Incubate 3 hours at room temperature. 14.3. Measure (1 hour) Prepare sufficient Assay Solution. Rinse wells twice with Buffer. Add 200 µl of Assay Solution to each well. Measure OD 450nm at approximately 1 minute intervals for 30 minutes. ab109721 Complex I Enzyme Activity Assay Kit 18

RESOURCES 15.TROUBLESHOOTING Problem Cause Solution Assay not working Use of ice-cold buffer Plate read at incorrect wavelength Buffers must be at room temperature Check the wavelength and filter settings of instrument Sample with erratic readings Lower/ Higher readings in samples and Standards Standard readings do not follow a linear pattern 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 Pipetting errors in standard or reaction mix Air bubbles formed in well Standard stock is at incorrect concentration Colorimetric: Clear plates Fluorometric: black wells/clear bottom plate Use deproteinization protocol provided 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 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 ab109721 Complex I Enzyme Activity Assay Kit 19

RESOURCES Problem Cause Solution Unanticipated results Measured at incorrect wavelength Check equipment and filter setting Samples contain interfering substances Sample readings above/ below the linear range Troubleshoot if it interferes with the kit Concentrate/ Dilute sample so it is within the linear range ab109721 Complex I Enzyme Activity Assay Kit 20

RESOURCES 16.INTERFERENCES These chemicals or biological materials will cause interferences in this assay causing compromised results or complete failure: RIPA buffer it contained SDS, which can destroy or decrease the activity of the enzyme. 17.FAQs How do I prepare my mitochondria samples? We have found that little or no optimization is necessary if crude mitochondria are made from samples. Mitochondria can be prepared by simple differential centrifugation of homogenized samples. How do I grow and prepare my cells? Complex I activity in cells from different origins differs greatly. Nontransformed fibroblasts have a higher activity than transformed cell lines such as HepG2 or HeLa cells. Consequently cell type and growth conditions are a large factor in Complex I activity measurement. Approximately how much protein is yielded from my plate of cells? We find the following typical yield of cells from a single confluent 177 cm 2 plate: Human fibroblasts: 1x10 7 cells - 1.5 mg total protein. Human HepG2: 2x10 7 cells - 3 mg total protein. It is recommended that you accurately determine the number of cells from your culture and the total protein yield. ab109721 Complex I Enzyme Activity Assay Kit 21

RESOURCES Can you recommend any positive controls? Any of the lysates mentioned below can be used as positive control in this assay: ab110338 Bovine Heart Mitochondrial lysate ab110346 Rat Liver Mitochondrial lysate ab110347 Rat Heart Mitochondrial lysate ab113048 Rat Brain Mitochondrial lysate ab110349 Mouse Liver Mitochondrial lysate ab110350 Mouse Heart Mitochondrial lysate ab110351 Mouse Brain Mitochondrial lysate ab109721 Complex I Enzyme Activity Assay Kit 22

RESOURCES 18.APPENDIX MITOCHONDRIAL PURIFICATION PROTOCOL Mitochondrial Purification Protocol Reagents needed: NKM buffer 1 mm Tris HCl, ph 7.4 0.13 M NaCl 5 mm KCl 7.5 mm MgCl 2 Homogenization buffer 10 mm Tris-HCl 10 mm KCl 0.15 mm MgCl 2 1 mm PMSF 1 mm DTT Always add PMSF and DTT immediately before use to NKM and homogenization buffer. Mitochondrial suspension buffer 10 mm Tris HCl, ph 6.7 0.15 mm MgCl 2 0.25 mm sucrose 1 mm PMSF ab109721 Complex I Enzyme Activity Assay Kit 23

RESOURCES Procedure 18.1. Collect cells by centrifugation at approximately 370 x g for 10 min. Decant supernatant and resuspend cells in 10 packed cell volumes of NKM buffer. 18.2. Pellet cells and decant supernatant, repeat this washing step two times. Resuspend cells in 6 packed cell volumes of homogenization buffer. 18.3. Transfer cells to a glass homogenizer and incubate for 10 min on ice. Using a tight pestle homogenize the cells. Check under the microscope for cell breakage, the optimum is around 60%. This may require 30 strokes or so of the pestle. 18.4. Pour homogenate into a conical centrifuge tube containing 1 packed cell volume of 2 M sucrose solution and mix gently. 18.5. Pellet unbroken cells, nuclei and large debris at 1,200 x g for 5 min and transfer the supernatant to another tube. This treatment is repeated twice, transferring the supernatant to a new tube each time, discarding the pellet. 18.6. Pellet the mitochondria by centrifuging at 7,000 x g for 10 min. Resuspend the mitochondrial pellet in 3 packed cell volumes of mitochondrial suspension buffer. Mitochondria are ready to use. ab109721 Complex I Enzyme Activity Assay Kit 24

RESOURCES 19.NOTES ab109721 Complex I Enzyme Activity Assay Kit 25

RESOURCES ab109721 Complex I Enzyme Activity Assay Kit 26

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