May 29, 2026
Verification of NAD(P)H Concentration by NanoDrop
- Daniel Moreira1
- 1i3S - Instituto de Investigação e Inovação em Saúde da Universidade do Porto
Protocol Citation: Daniel Moreira 2026. Verification of NAD(P)H Concentration by NanoDrop. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jjq5vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 18, 2026
Last Modified: May 29, 2026
Protocol Integer ID: 317324
Keywords: enzymatic activity, uv-vis, NADH, NADPH, spectrophotometer, kinetics, extinction coefficient, absorptivity coefficient, spectrophotometry, absorbance at 340 nm, NanoDrop, cofactor quantification, reduced pyridine nucleotides, Beer–Lambert law, enzymatic assays, cofactor stability, redox cofactors, concentration determination, nadph solution, based spectrophotometric measurement, spectrophotometric measurement, nadph, enzymatic assay, reduced pyridine nucleotide, nanodrop, vis spectrophotometer, pyridine nucleotide
Disclaimer
If the measured NAD(P)H concentration is substantially lower than expected, it is recommended to prepare a fresh solution rather than adjusting volumes to reach the target final concentration. This is because degraded samples may contain a significant proportion of oxidized forms (NAD⁺/NADP⁺), which can persist in the mixture and potentially interfere with downstream enzymatic assays, including by inhibiting enzyme activity.
Abstract
Both NADH and NADPH exhibit strong absorbance at 340 nm, whereas their oxidized forms (NAD⁺ and NADP⁺) do not. Their concentration can therefore be determined directly from the absorbance of the reduced form using the Beer–Lambert law, without the need for a standard curve. Because the NanoDrop automatically normalizes measurements to a 1 cm path length equivalent, the calculation is identical to that used for conventional cuvette-based spectrophotometric measurements. Although this method cannot distinguish between NADH and NADPH, it provides a rapid and practical means of verifying the concentration of either NADH or NADPH solutions prior to enzymatic assays. This is particularly important because the activity of many NAD(P)H-dependent enzymes is highly sensitive to their concentration, and reduced pyridine nucleotides are prone to degradation in aqueous solution during storage and handling. The protocol can also be used to verify NAD(P)H concentration using conventional cuvette UV-Vis spectrophotometers.
Materials
- NADH or NADPH stock solution (prepared at 5 mM in ultrapure water or 1× PBS)
- Matching blank (same solvent as the stock)
- NanoDrop spectrophotometer
- Lint-free wipes (e.g., KimWipes)
Before start
Tip: Re-blank between samples if the solvent changes. Re-blank after prolonged interruptions (> 15 min) to account for pedestal drift.
Tip: NADH and NADPH degrade rapidly at room temperature. Keep stocks on ice, avoid repeated freeze–thaw cycles, and measure promptly after thawing. Prepare fresh working solutions or freshly thawed frozen aliquots.
Tip: The blank must exactly match the solvent in which the stock is dissolved (ultrapure water or 1× PBS). Components that absorb at 340 nm will cause overestimation.
Procedure
Open the NanoDrop software and select UV-Vis or the custom absorbance measurement mode.
Set the measurement wavelength to 340 nm and the extinction coefficient to 6.22 mM−1·cm−1 if the software prompts for it.
Clean the upper and lower pedestals with a dry lint-free wipe.
Pipette 1–2 µL of blank solvent onto the lower pedestal and close the arm.
Select Blank.
Wipe both pedestals clean afterwards.
If the stock concentration is ≥ 1 mM, prepare a diluted working sample (e.g., 1:20–1:100 in blank solvent) before measuring.
Pipette 1–2 µL of sample (or diluted sample) onto the lower pedestal and close the arm.
Select Measure.
Record the A340 and the calculated concentration.
Wipe both pedestals clean and repeat Step 6 for a total of 3 independent readings.
Register the mean ± SD of the three measurements.
Calculation
Calculate the stock concentration using the formula:
Example - A340 = 0.245 for a sample diluted 1:100 from the stock
Record the following in your lab notebook:
- Date of measurement
- Nucleotide identity (NADH or NADPH) and preparation date
- Solvent used for blank
- Dilution factor applied before measurement
- Three A340 readings and calculated stock concentrations
- Mean ± SD
- Any deviations from this protocol