How to Detect NADH

Nicotinamide adenine dinuceltoide (NADH), abbreviated "NAD", is a coenzyme found in living cells that produces chemical reactions in proteins. Pharmaceutical companies study NADH because of its metabolism process, and synthetically create it for a number of purposes. Since NADH is microscopic, the naked eye cannot spot it. You need scientific testing and an assay kit to detect its presence.

Things You'll Need

• Dry ice
• Micro-centrifuge
• Eppendorf tubes

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Instructions

Reagent Reconstitution

• 1

  Set the cycling buffer on a counter and allow the buffer to reach room temperature. The ideal temperature for the buffer is 22 degrees Celsius.

• 2

  Reconstitute the NAD cycling enzyme mix with exactly 220 microliters of cycling buffer. Freeze the solution in temperatures beneath -70 degrees Celsius.

• 3

  Reconstitute the NADH developer with 1.2 ml of ddH2O. Gently mix the solution until it is completely dissolved. Do not vortex.

• 4

  Reconstitute NADH standard with 200 microliters of pure dimethyl sulfoxide.

Sample Preparation

• 5

  Wash the cells with phosphate-buffered saline.

• 6

  Pellet 2x10(5) cells for each individual assay into a micro-centrifuge tube and run at 2,000 RPM for 5 minutes.

• 7

  Extract the cells with 400 microliters of NAD/NADH extraction buffer by freezing and thawing the cells in two cycles--20 minutes on dry ice and 10 minutes at room temperature. Vortex the extracted cells for exactly 10 seconds.

• 8

  Spin the cell samples in a micro-centrifuge at 14,000 RPM for exactly 5 minutes.

• 9

  Transfer the NAD/NADH cells into a clean tube.

Assay Protocol

• 10

  Dilute 10 microliters of the NADH standard with 990 microliters NAD/NADH extraction buffer. Add 0, 2, 4, 6, 8, 10 microliters of the diluted solution into a 96-well plate in duplicate to create 0, 20, 40, 60 80, 100 well standard. Fill the last well with 50 microliters of the NAD/NADH extraction buffer.

• 11

  Transfer 50 microliters of the extracted cell samples into a 96-well plate in duplicates as before.

• 12

  Prepare the cell samples for NADH detection. Decompose the NAD from the cell samples by putting 200 microliters of the extracted cell samples into eppendorf tubes. Heat the tubes to 60 degrees celsius for exactly 30 minutes into a temperature controlled water bath. Quickly cool the samples by placing the tubes on ice. Spin the samples in the micro-centrifuge to remove precipitates. Transfer 50 microliters of the decomposed samples into a 96-well plate in duplicates as before.

• 13

  Mix the NAD cycling buffer mix with 100 microliters of NAD cycling buffer mix and 2 microliters of NAD cycling enzyme. Add 100 microliters of this new solution into each well of NADH standards and samples.

• 14

  Incubate the plate at room temperature for exactly 5 minutes. This process will convert the NAD to NADH.

• 15

  Add 10 microliters of NADH developer into every well. Allow the plate to sit at room temperature for a minimum of 1 hour and a maximum of 4 hours.

• 16

  Read the plate and calculate the NADH.