How to Measure NADH
NAD and NADH are both pyridine nucleotides involved in metabolism. NADH and NAD are co-enzymes found in the mitochondria, the powerhouses of the cell. Both NAD and NADH are involved in cellular reactions such as respiration and photosynthesis where energy and hydrogen are generated. NAD and NADH fuel biochemical reactions which require a number of enzymes to produce ATP, the main energy source in cells.
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Things You'll Need
- Cells or tissue under study
- NADH assay kit
- Phosphate-buffered saline
- Dry Ice
- Heating block
- 96 well plate
- Centrifuge
- Labeled tubes
- Plate reader
- Vortex
- Known concentration of NADH
Instructions
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Assay
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1
Wash the cells or tissue under study with cold phosphate-buffered saline, PBS. Extract cells or break up tissue in extraction buffer by placing the mixture on dry ice for 20 minutes and then at room temperature for 10 minutes. Repeat.
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2
Vortex for 10 seconds. Spin in a centrifuge for 5 minutes to pellet cell or tissue debris. Remove supernatant, the liquid phase, and place the liquid in a clean tube.
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3
Filter supernatant through a specialized filter to remove enzymes that will breakdown NADH.
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4
Remove 200 micro liters, place in a clean tube and heat at 60 degrees Celsius in a heating block for 30 minutes to denature NAD. Cool and centrifuge and remove liquid phase. Transfer 50 micro liters into a 96-well plate; each sample should be in duplicate or triplicate. To determine total NAD, NADt, do not heat.
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5
Prepare and add cycling mix to the 96-well plate. Mix and incubate for 5 minutes. Add 10 micro liters of developer and leave to incubate at room temperature for up to 4 hours. Add stop solution. Read color change on a plate reader or fluorimeter depending on kit.
Standard Curve and Calculation
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6
Prepare a standard curve by taking a known concentration of NADH and diluting it in extraction buffer. Dilute into different concentrations, ensuring the end volume remains the same as test samples. Plate on the same 96-well plate as test samples. Read optical density.
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7
Draw standard curve graph with concentration on the X axis and optical density on the Y axis. Take an average of each test sample and read the concentration from the standard curve graph.
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8
Calculate the NAD/NADH ratio by subtracting total NAD, NADt, from NADH and then dividing by NADH.
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1
Tips & Warnings
A number of commercially available assay kits are available.
References
- Photo Credit laboratory image by Oleg Verbitsky from Fotolia.com
