Mitochondrial Complex I Activity Assay Kit

User Protocol TB554 Rev.
B 0612JN
Page 1 of 9
Mitochondrial Complex I Activity Assay Kit

Table of Contents
About the Kits......................................................................................................... 2
Description
Components
Storage
Materials Required But Not Supplied
2
2
2
2
Assay Preparation.................................................................................................. 3
Considerations Before You Begin
Buffer and Sample Preparation
3
3
Assay Protocol ....................................................................................................... 4

Plate Loading
Measurement
4
4
Performance Characteristics ................................................................................. 5

Appendix A: Assay Flow Chart .............................................................................. 7
Appendix B: Frequently Asked Questions ............................................................. 8
Appendix C: Assay Plate Design ........................................................................... 9
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FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
Cat. No.
User Protocol TB554 Rev. B 0612JN
Page 2 of 9Page
About the Kits

Mitochondrial Complex I Activity Assay Kit
AAMT001-1KIT
Description
The Mitochondrial Complex I Activity Assay Kit (Cat. No. AAMT001-1KIT) is used to determine the activity of
mitochondrial OXPHOS Complex I (NADH dehydrogenase, E.C. 1.6.5.3). The enzyme complex is immunocaptured
within the wells of the microplate and activity is determined by following the oxidation of NADH to NAD+ and the
simultaneous reduction of a dye which leads to increased absorbance at 450 nm.
NADH
NAD+
H
Dye
Increase Abs.450nm
Complex I activity reaction scheme. The oxidation of NADH is linked to the reduction of a dye,
which has increased absorbance at 450 nm and so appears yellow in color.
Components
25 ml
10 ml
1 ml
1 ea
1 ea
1 ea
Buffer, (72560-25ML)
10X Blocking Solution, (72561-10ML)
Detergent, (72562-1ML)
20X NADH (40 mM), (72563-1EA)*
100X Dye, (72564-1EA)**
Microplate, (72565-1EA)
This kit contains sufficient materials for 96 measurements (one 96-well microplate).
* Add 1.1 ml of dH20. Then, follow the storage instructions below.
** Add 0.25 ml of dH20. Then, follow the storage instructions below.
Note: The 96-well microplate has a monoclonal antibody pre-bound to the wells. This plate can be separated into twelve
8-well strips and thus can be used for up to 12 individual experiments.
Storage
Store all components at 4C. After hydration of the 20X NADH and 100X Dye with dH20, both of these components
must be stored at 80C and are stable for at least six months from date of receipt. It is also recommended to aliquot them
before freezing to prevent repeated freeze thaw cycles.
Materials Required But Not Supplied
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Spectrophotometer that measures absorbance at 450 nm

Deionized water
Multichannel pipette
Protein assay method
PBS (1.4 mM KH2P04, 8 mM Na2HP04, 140 mM NaCl, 2.7 mM KCl, pH 7.3)
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Page 3 of 9Page
Assay Preparation
Considerations Before You Begin
This protocol contains detailed steps for measuring the activity of mitochondrial OXPHOS Complex 1. Be completely
familiar with the protocol and the FAQ section before beginning the assay. Do not deviate from the specified protocol
steps or optimal results may not be obtained.
This rapid Complex I microplate has been developed for use with human, rat and mouse samples (bovine
samples are also compatible). Samples may be prepared as mitochondria, crude mitochondria and in most cases
whole tissue or cell lysates are suitable but may require some sample optimization.
The protein concentration of the sample should be measured. Yields of protein / cell numbers from cell culture pellets
are suggested in frequently asked questions. Once diluted to the specified concentration the sample is detergent
solubilized and diluted to within the linear range of measurement. If you are performing mitochondrial toxicity testing,
an untreated control or normal sample should always be included in the assay as a reference measurement. In addition,
a buffer control should be used as a negative control.
Table 1. Typical ranges of measurement per 200 l well volume
Sample Type
Typical Linear Range
Tissue mitochondria extract
5100 g
Cultured cell extract*
25250 g
*Note: Complex I 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.
Note that this activity assay measures the diaphorase-type 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 the activity assay is affected by assembly deficiencies.
Typical intra-assay variations (same day, same sample) < 10% (n = 60), inter-assay variation < 15% (n = 60).
Buffer and Sample Preparation

1.
Add contents of 20X Buffer (25 ml) to 475 ml deionized H2O. Label this mixture as Buffer. Mix well.
2.
Pour off 90 ml of Buffer and add to this amount 10 ml of Blocking Solution. Label this Incubation Solution.
3.
Determine the sample protein concentration using a standard method such as BCA method (Pierce). Adjust the
concentration of the sample to 5.5 mg/ml using PBS.
4.
For details of sample solubilization conditions see frequently asked questions section.
5.
Extract the proteins by adding Detergent. To do this add 1/10 of a volume of Detergent (e.g. if the total sample
volume is 500 l, add 50 l of Detergent). Mix well. Place the tube on ice for 30 minutes to allow solubilization.
Note the sample concentration is now 5 mg/ml.
6.
Centrifuge the sample for 20 minutes at approximately 12,000 x g for 20 minutes. Collect the supernatant.
7.
Dilute all samples to the desired concentration in Incubation Solution. Table 1 above shows a good linear range for
the assay, Table 2 below shows a recommended mid-range value at which to load an experimental sample.
Table 2. Recommended sample dilutions per well (200 l)
Sample Type
Tissue mitochondria extract
Heart
Liver
Cultured cell extracts
Fibroblast (MRC5)
Hepatoblastoma (HepG2)
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Recommended Amount
20 g
50 g
100 g
200 g

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Assay Protocol
Plate Loading
1.
Add 200 l of each diluted sample into each well of the microplate that will be used for this experiment. Include a
normal sample as a positive control. Also include a buffer control (50 l of Solution 1) as a null or background
reference.
2.
Incubate for 3 hours at room temperature.
Measurement
Note: For multiple experiments 20X NADH and 100X Dye should be aliquotted and stored at 80C according to frequently
asked questions.
1.
Thaw 20X NADH and 100X Dye.
2.
Empty the wells by turning the plate over and shaking out any remaining liquid. Blot the plate face down on paper
towel. Add 300 l of Buffer.
3.
Make Assay Solution (1.75 ml/strip) according to the following table:

No. of Strips
Buffer (ml)
20X NADH (l)
100X Dye (l)
1
2
3
1.67
3.33
5.00
84
167
250
17
33
50
6.67
333
67
5
6
7
8
9
10
11
8.33
10.0
11.67
13.33
15.00
16.67
18.33
417
500
583
667
750
833
917
83
100
117
133
150
167
183
12
20.00
1000
200
4.
Empty the wells of the microplate as above.
5.
Rinse all wells once more with 300 l Buffer.
6.
Empty the wells again.
7.
Add 200 l of Assay Solution to each well carefully to avoid bubbles. Any bubbles should be popped with a fine
needle as rapidly as possible.
8.
Place the plate in the reader and record with the following kinetic program.
9.
Save data and analyze as described in the Performance Characteristics section.

Kinetic Measurement
OD 450 nm
Time: 30 minutes
Interval: 2060 seconds
Temperature: Room Temperature
Autoshake between readings
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Performance Characteristics
Note: All data and results shown below are representative and not lot specific.
Complex I activity in each well is proportional to the increase in absorbance at 450 nm within each well. The activity is
expressed as the change in absorbance per minute per amount of sample loaded into the well.
Examine the linear rate of increase in absorbance at 450 nm over time. An example is shown below where the
rate/slope is calculated between these time points. Most microplate software is capable of performing this function.
Repeat this for all samples.
Raw data - change OD 450 nm:
This raw data can be expressed as rate (mOD/min) per g of cell lysate added per well as shown below.
Range of assay:
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As examples: (A) the assay was used to determine the Complex I activity in normal fibroblasts and Rho0 fibroblasts
(cells in which the mtDNA is removed and hence essential Complex I proteins are not expressed). The Rho0 cells
showed no/little complex I activity. In a similar analysis (B) rat cardiomyocytes were grown for 5 days in 40 M
chloramphenicol (CAM) to inhibit mitochondrial protein synthesis, Complex I assembly and hence activity was greatly
reduced:
(A) Human fibroblasts and Rho0

fibroblasts:
(B) Rat cardiomyocytes treated with 40 M

chloramphenicol (CAM)
This assay measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples
within the recommended ranges given on page 3. Note that these ranges depend on mitochondria preparation quality.
Examples of Complex I activity measured in different rat and mouse mitochondrial samples are shown below:
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Appendix A: Assay Flow Chart

Note: For quick reference only. Be completely familiar with the previous details of this protocol before performing the assay.
Prepare Sample (approximately 1 hour)
Adjust sample concentration to 5.5 mg/ml in PBS.

Perform Detergent extraction with 1/10 volume Detergent.
Incubate on ice for 30 minutes.
Centrifuge at 12,000 x g for 20 minutes and then collect supernatant.
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.
Measure (0.5 hours)
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Make sufficient Assay Solution.

Rinse wells twice with Buffer.
Add 200 l of Assay Solution to each well.
Measure OD450 at approximately 1 minute intervals for 30 minutes.
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Page 8 of 9Page
Appendix B: Frequently Asked Questions

1.
How should I store the kit?

Wash Buffer, 10X Blocking Solution, the covered microplate and the detergent should be refrigerated and are stable
for at least 6 months. The diluted Buffer can be stored at room temperature; however for extended periods of time
( > 1 month), it should be stored in the refrigerator to prevent microbiological growth.
2.
How should I store 20X NADH and 100X Dye?

Both reagents are temperature sensitive. Upon receipt, both components may be stored at 4C prior to hydration
with dH20. After hydration, they must be stored at 80C and are stable for at least six months from date of receipt.
It is also advisable to aliquot them before freezing to prevent repeated freeze thaw cycles.
3.
How should I aliquot?

Divide 20X NADH and 100X Dye into aliquots depending on how many experiments you wish to run (see table on
page 4). The plate comes in 12 strips, therefore it is anticipated that up to 12 experiments on different days could be
done. Because the 20X NADH is hydrated with 1.1 ml of dH20, you could further aliquot each tube into 12 x 91 l
aliquots and immediately freeze at 80C for storage until use. Similarly, the 100X Dye could be aliquoted into
12 separate 18 l aliquots each.
4.
How do I prepare mitochondria samples?

Little or no optimization is necessary if crude mitochondria are made from samples. Mitochondria can be prepared
by simple differential centrifugation of homogenized samples. For more information, use a general mitochondria
isolation protocol as found in literature.
5.
How do I grow and prepare cultured cell samples?

Complex I activity in cells from different origins differs greatly. Non-transformed 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.
6.
Approximately how much protein is yielded from my plate of cells?

We find the following typical yield of cells from a single confluent 177 cm2 plate:
Sample Type
Number of Cells
Amount of Protein
Human fibroblasts
1x10 cells
1.5 mg total protein
Human HepG2
2x107 cells
3 mg total protein
It is recommended that to accurately determine the number of cells and the total yield protein from the first
confluent plate.
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Page 9 of 9Page
Appendix C: Assay Plate Design
3
4
5
6
7
8
9
10 11 12
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____ ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ ____
2
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User Protocol TB554 Rev.

Mitochondrial Complex I Activity Assay Kit

Assay Protocol ....................................................................................................... 4

Performance Characteristics ................................................................................. 5

USA and Canada

All Other Countries

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

User Protocol TB554 Rev. B 0612JN

About the Kits

Materials Required But Not Supplied

USA and Canada

Spectrophotometer that measures absorbance at 450 nm

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

Typical Linear Range

Tissue mitochondria extract

Cultured cell extract*

Buffer and Sample Preparation

USA and Canada

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

Incubate for 3 hours at room temperature.

Thaw 20X NADH and 100X Dye.

Make Assay Solution (1.75 ml/strip) according to the following table:

20X NADH (l)

100X Dye (l)

Empty the wells of the microplate as above.

Rinse all wells once more with 300 l Buffer.

Empty the wells again.

Save data and analyze as described in the Performance Characteristics section.

USA and Canada

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

Raw data - change OD 450 nm:

USA and Canada

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

(A) Human fibroblasts and Rho0

(B) Rat cardiomyocytes treated with 40 M

USA and Canada

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

Appendix A: Assay Flow Chart

Prepare Sample (approximately 1 hour)

Adjust sample concentration to 5.5 mg/ml in PBS.

Load Plate (3 hours)

Measure (0.5 hours)

USA and Canada

Make sufficient Assay Solution.

United Kingdom and Ireland

All Other Countries

User Protocol TB554 Rev. B 0612JN

Appendix B: Frequently Asked Questions

How should I store the kit?

How should I store 20X NADH and 100X Dye?

How should I aliquot?

How do I prepare mitochondria samples?

How do I grow and prepare cultured cell samples?

Approximately how much protein is yielded from my plate of cells?