Version: 5 Last Updated: 26 April 2016

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Table of Contents
1.
Overview
2.
Protocol Summary
3.
Kit Components
4.
Storage and Stability
5.
Materials Required, Not Supplied
6.
Assay Protocol
7.
Data Analysis
8.
Troubleshooting
8
13
15
7
7
5
6
2
3
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Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are two important cofactors found in cells. NADH is the reduced form of NAD+, and
NAD+ is the oxidized form of NADH. NAD forms NADP with the addition of a phosphate group to the 2' position of the adenyl nucleotide through an ester linkage. NADP is used in anabolic biological reactions, such as fatty acid and nucleic acid synthesis, which require NADPH as a reducing agent. In chloroplasts, NADP is an oxidizing agent important in the preliminary reactions of photosynthesis. The NADPH produced by photosynthesis is used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. The traditional NAD/NADH and NADP/NADPH assays are done by monitoring the changes in NADH or NADPH absorption at 340 nm. This method suffers low sensitivity and high interference since the assay is done in the UV range that requires expensive quartz microplates.
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Abcam's NAD/NADH Assay Kit (Fluorometric) (ab176723) provides a convenient method for sensitive detection of NAD, NADH and their ratio. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interference from biological samples. There is no need to purify
NAD/NADH from sample mix. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em =
530 - 570/590 - 600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm. This kit provides NAD and NADH extraction buffer, and cell lysis buffer for your convenience. It has been frequently used for determining
NAD/NADH from cell lysates.
Kit Key Features
 Broad Application – can be used for quantifying
NAD/NADH in solutions and in cell extracts
 Sensitive – detect as low as 10 picomoles of NAD/NADH in solution
 Continuous – suitable for both manual and automated operations without a mixing or separation step
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 Convenient – formulated to have minimal hands-on-time
 Non-radioactive – no special requirements for waste treatment
Prepare NADH standards and/or test samples
Add NADH or NAD Extraction Solution
Incubate at RT for 15 min
Add NAD or NADH Extraction Solution
Add NAD/NADH reaction mixture
Incubate at RT for 15 min to 2 hours
Monitor fluorescence intensity at Ex/Em =540/590 nm
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Item Quantity Storage upon arrival
Storage after use/ reconstituti on
-20°C -20°C NAD/NADH Recycling
Enzyme Mixture (lyophilized powder)
NADH Sensor Buffer
NADH Standard (142 µg)
NADH Extraction Solution
NAD Extraction Solution
NAD/NADH Control Solution
NAD/NADH Lysis Buffer
2 bottles
20 mL
1 vial
10 mL
10 mL
10 mL
10 mL
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
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Upon arrival, store the kit at-20°C and protected from light. Please read the entire protocol before performing the assay. Avoid repeated freeze/thaw cycles.
Warm all buffers to room temperature before use. Briefly centrifuge all small vials prior to opening.
 96 or 384-well black plate with clear flat bottoms
 Multi-well spectrophotometer (ELISA reader)
 Distilled water (ddH
2
O)or MilliQ
 PBS
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1. Reagent Preparation a) NADH Stock Solution:
Add 200 µL of PBS buffer into the vial of NADH standard to have
1 mM (1 nmol/µL) NADH stock solution.
NOTE: The unused NADH stock solution should be divided into single use aliquots and stored at -20 o C.
b) NAD/NADH reaction mixture:
Add 10 mL of NADH Sensor Buffer to the bottle of NAD/NADH
Recycling Enzyme Mixture and mix well.
NOTE: This NAD/NADH reaction mixture is enough for two 96well plates. The unused NAD/NADH reaction mixture should be divided into single use aliquots and stored at -20 o C.
2. Sample preparation a) For lysis of plant cells:
Homogenize the leaves with the lysis buffer at 200 mg/mL, and then centrifuge at 2500 rpm for 5-10 minutes, use the supernatant for the assay. Lyse cells by resuspending them in 1 mL ice cold lysis buffer and pipetting up and down. Incubate on ice for 5 min. Centrifuge samples at maximum speed for 10 min in a cold centrifuge and collect supernatant.
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b) For lysis of bacterial cells:
Collect bacterial cells by centrifugation ((10,000 x g, 0°C, 15 min).
Use about 100 to 10 million cells/mL lysis buffer, leave at room temperature for 15 minutes. And then centrifuge at 2500 rpm for 5 minutes, use the supernatant for the assay.
c) For lysis of mammalian cells:
Simply remove medium from the plates (wells), use about 100 uL lysis buffer per 1-5 million cells (or 100uL/ well in a 96-well cell culture plate), and leave at room temperature for 15 minutes. You can use the cell lysate directly or simply centrifuge at 1500 rpm for 5 minutes, use the supernatant for the assay.
d) For lysis of tissues:
Weigh ~20 mg tissue, wash with cold PBS, homogenize with 400 μL of lysis buffer in a micro-centrifuge tube, and then centrifuge at 2500 rpm for 5-10 minutes, use the supernatant for the assay.
3. Standard Curve Preparation: a) Add 30 µL of 1 mM NADH stock solution (from Step 1a) into
970 µL PBS buffer (pH 7.4) to generate 30 µM
(30 pmols/µL) NADH standard solution.
NOTE: Diluted NADH standard solution is unstable, and should be used within 4 hours.
b) Take 200 µL of 30 µM NADH standard solution (from Step
3a) and perform 1:3 serial dilutions in PBS to get 10, 3.3,
1.1, 0.37, 0.12, 0.04 and 0 µM serially diluted NADH standards.
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c) Add serially diluted NADH standards and/or NAD/NADH containing test samples into a solid black 96-well microplate, as described in the tables below.
NOTE: Prepare cells or tissue samples as desired. NAD/NADH
Lysis Buffer can be used for lysing the cells for convenience.
NS
3
NS
4
NS
5
NS
6
NS
7
BL BL TS TS TS
(NADH)
NS
1
NS
1
….
….
….
NS
2
NS
2
NS
3
NS
4
NS
5
NS
6
NS
7
TS
(NADH)
….
TS
(NAD)
TS
(NAD)
NOTE: NS = NADH Standards, BL = Blank Control, TS = Test
Samples; TS (NADH) = Test samples treated with NADH Extraction
Solution for 10-15 minutes, then neutralized by NAD Extraction solution; TS (NAD) = Test Samples treated with NAD Extraction
Solution for 10-15 minutes, then neutralized by NADH Extraction
Solution.
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NADH
Standard
Serial
Dilutions*:
25 μL
NAD/NADH
Control
Solution:
25 μL
Blank
Control
PBS: 25 μL
NAD/NADH
Control
Solution:
25 μL
Test Sample
(NAD/NADH)
Test Sample:
25 μL
NAD/NADH
Control
Solution:
25 μL
Test
Sample
(NADH
Extract)
Test
Sample:
25 μL
NADH
Extraction
Solution:
25 μL
Test
Sample
(NAD
Extract)
Test
Sample: 25
μL
NAD
Extraction
Solution:
25 μL
NAD/NADH
Control
Solution:
25 μL
Incubate at room temperature for 10 to 15 minutes
NAD/NADH
Control
Solution:
25 μL
Total: 75 μL Total: 75 μL
NAD/NADH
Control
Solution:
25 μL
NAD
Extraction
Solution:
25 μL
NADH
Extraction
Solution:
25 μL
Total: 75 μL Total: 75 μL Total: 75 μL
*Note: Add the serially diluted NADH standards from 0.03 μM to
30 μM into wells from NS1 to NS7 in duplicate. High concentration of
NADH (e.g., > 300 μM, final concentration) may cause reduced fluorescence signal due to the over oxidation of NADH sensor (to a non-fluorescent product). d) For NADH Extraction (NADH): Add 25 μL of NADH
Extraction Solution into the wells of NAD/NADH containing test samples. Incubate at room temperature for 10 to
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15 minutes, then, add 25 μL of NAD Extraction Solution to neutralize the NADH extracts.
e) For NAD Extraction (NAD): Add 25 μL of NAD Extraction
Solution into the wells of NAD/NADH containing test samples. Incubate at room temperature for 10 to
15 minutes, then, add 25 μL of NADH Extraction Solution to neutralize the NAD extracts.
f) For Total NAD and NADH: Add 25 μL of NAD/NADH
Control Solution into the wells of NADH standards and
NAD/NADH containing test samples. Incubate at room temperature for 10 to 15 minutes, and then add 25 μL of
Control Solution.
4. Run NAD/NADH assay in supernatants reaction: a) Add 75 µL of NADH reaction mixture into each well of
NADH standard, blank control, and test samples to make the total NADH assay volume of 150 µL/well.
b) Incubate the reaction at room temperature for 15 minutes to
2 hours, protected from light.
c) Measure the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm.
NOTE: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 +/- 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.
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The fluorescence in blank wells (with the PBS buffer only) is used as a control, and is subtracted from the values for those wells with the
NADH reactions. The typical data are shown in Figure 1 (Total NAD and NADH vs. NAD or NADH Extract).
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Figure 1.
Total NADH and NAD, and their extract dose response were measured with Abcam’s NAD/NADH Assay Kit (Fluorometric) in a 96-well black plate using a Gemini microplate reader. 25 µL of equal amount of NAD and NADH was treated with or without NADH or NAD extraction solution for 15 minutes, and then neutralized with extraction solutions at room temperature. The signal was acquired at
Ex/Em = 540/590 nm (cut off at 570 nm) 30 minutes after adding
75 µL of NADH reaction mixture. The blank signal was subtracted from the values for those wells with the NADH reactions (Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point).
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Problem
Assay not working
Reason
Assay buffer at wrong temperature
Protocol step missed
Plate read at incorrect wavelength
Unsuitable microtiter plate for assay
Unexpected results
Measured at wrong wavelength
Samples contain impeding substances
Unsuitable sample type
Sample readings are outside linear range
Solution
Assay buffer must not be chilled
- needs to be at RT
Re-read and follow the protocol exactly
Ensure you are using appropriate reader and filter settings (refer to datasheet)
Fluorescence: Black plates
(clear bottoms);
Luminescence: White plates;
Colorimetry: Clear plates.
If critical, datasheet will indicate whether to use flat- or U-shaped wells
Use appropriate reader and filter settings described in datasheet
Troubleshoot and also consider deproteinizing samples
Use recommended samples types as listed on the datasheet
Concentrate/ dilute samples to be in linear range
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Problem
Samples with inconsistent readings
Reason
Unsuitable sample type
Samples prepared in the wrong buffer
Samples not deproteinized (if indicated on datasheet)
Cell/ tissue samples not sufficiently homogenized
Too many freezethaw cycles
Samples contain impeding substances
Samples are too old or incorrectly stored
Lower/
Higher readings in samples and standards
Not fully thawed kit components
Out-of-date kit or incorrectly stored reagents
Reagents sitting for extended periods on ice
Incorrect incubation time/ temperature
Incorrect amounts used
Solution
Refer to datasheet for details about incompatible samples
Use the assay buffer provided
(or refer to datasheet for instructions)
Use the 10kDa spin column
(ab93349)
Increase sonication time/ number of strokes with the
Dounce homogenizer
Aliquot samples to reduce the number of freeze-thaw cycles
Troubleshoot and also consider deproteinizing samples
Use freshly made samples and store at recommended temperature until use
Wait for components to thaw completely and gently mix prior use
Always check expiry date and store kit components as recommended on the datasheet
Try to prepare a fresh reaction mix prior to each use
Refer to datasheet for recommended incubation time and/ or temperature
Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume)
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Problem
Standard curve is not linear
Reason
Not fully thawed kit components
Pipetting errors when setting up the standard curve
Incorrect pipetting when preparing the reaction mix
Solution
Wait for components to thaw completely and gently mix prior use
Try not to pipette too small volumes
Always prepare a master mix
Air bubbles in wells
Concentration of standard stock incorrect
Errors in standard curve calculations
Use of other reagents than those provided with the kit
Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates
Recheck datasheet for recommended concentrations of standard stocks
Refer to datasheet and re-check the calculations
Use fresh components from the same kit
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All information / detail is correct at time of going to print.