ab65348 NAD/NADH Assay Kit - Abcam

ab65348
NAD/NADH Assay Kit
Instructions for Use
For the rapid, sensitive and accurate
measurement of NAD/NADH in various samples.
This product is for research use only and is not
intended for diagnostic use.

Table of Contents
1. Overview 3
2. Protocol Summary 4
3. Components and Storage 5
4. Assay Protocol 7
5. Data Analysis 10
6. Troubleshooting 11
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1. Overview
Assay of nicotinamide nucleotides is of continual interest in the
studies of energy transforming and redox state of cells or tissues.
Abcam’s NADH/NAD Quantification Kit provides a convenient tool for
sensitive detection of the intracellular nucleotides: NADH, NAD and
their ratio. The NAD Cycling Enzyme Mix in the kit specifically
recognizes NADH/NAD in an enzyme cycling reaction. There is no
requirement to purify NADH/NAD from samples.
The reaction specifically detects NADH and NAD, but not NADP nor
NADPH. The enzyme cycling reaction significantly increases the
detection sensitivity and specificity. NADt (NAD and NADH) or
NADH can be easily quantified by comparing with standard NADH.
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2. Protocol Summary
Sample Preparation
Standard Curve Preparation
Prepare and Add Reaction Mix
Measure Optical Density
4

3. Components and Storage
A. Kit Components
Item
Quantity
NADH/NAD Extraction Buffer
50 mL
NAD Cycling Buffer
15 mL
NAD Cycling Enzyme Mix
1 vial
NADH Developer
1 vial
Stop Solution
1.2mL
NADH Standard (MW:763) 152.6 µg
Store kit at -20°C.
The enzymes are stable for up to 2 months at -80°C after
reconstitution.
Ensure that the NAD Cycling Buffer is at room temperature before
use. Keep other enzymes on ice during the assay and protect from
light.
5

B. Additional Materials Required
• Microcentrifuge
• Pipettes and pipette tips
• Colorimetric microplate reader
• 96 well plate
• Orbital shaker
6

4. Assay Protocol
1. Sample Preparation:
a) For cell samples: Wash cells with cold PBS. Pellet 2 X 10 5 cells
for each assay in a micro-centrifuge tube (2000 rpm for 5 min).
Extract the cells with 400 µl of NADH/NAD Extraction Buffer by
freeze/thaw two cycles (20 min on dry-ice, then 10 min at room
temperature), or homogenization. Vortex the extraction for
10 sec. Spin the sample at 14000 rpm for 5 min. Transfer the
extracted NADH/NAD supernatant into a labeled tube.
b) For tissue samples (weight ~20 mg): Wash with cold PBS,
homogenize with 400 µl of NADH/NAD Extraction Buffer in a
micro-centrifuge tube. Spin the sample at 14000 rpm for 5 min.
Transfer the extracted NADH/NAD supernatant into a new tube.
Note:
Cell or tissue lysates may contain enzymes that consume NADH
rapidly. We suggest removal of these enzymes by filtering the
samples through 10 kDa molecular weight cut off filters (ab93349)
before performing the assay.
2. Standard Curve Preparation:
Reconstitute NADH standard with 200 µl of pure DMSO to generate
1 nmol/µl NADH standard solution. Dilute 10 µl of the 1 nmol/µl
NADH standard with 990 µl NADH/NAD Extraction Buffer to
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generate 10 pmol/µl standard NADH (Note: diluted NADH solution is
unstable, must be used within 4 hours).
Add 0, 2, 4, 6, 8, 10 µl of the diluted NADH standard into labeled
96-well plate in duplicate to generate 0, 20, 40, 60, 80, 100 pmol/well
standard. Make the final volume to 50 µl with NADH/NAD extraction
buffer.
a. To detect total NADt (NADH and NAD): Transfer 50 µl of
extracted samples into labeled 96-well plate in duplicates.
We recommend performing several different sample dilutions with
the Extraction Buffer to ensure the readings fall in the standard
curve range.
b. To detect NADH: NAD needs to be decomposed before the
reaction. To decompose NAD, aliquot 200 µl the extracted
samples into eppendorf tubes. Heat to 60°C for 30 min in a water
bath or a heating block. Under these conditions, all NAD will be
decomposed, while NADH will still be intact. Cool samples on ice.
Quick spin the samples to remove precipitates if precipitation
occurs.
Transfer 50 µl of NAD decomposed samples into labeled 96-well
plate in duplicates.
We recommend performing several different sample dilutions with
the Extraction Buffer to ensure the readings fall in the standard
curve range.
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3. NAD Cycling Mix:
a) Reconstitute NAD Cycling Enzyme Mix with 220 µl NAD Cycling
Buffer. Reconstitute NADH developer with 1.2 ml of ddH 2 O.
Pipette up and down several times to completely dissolve the
pellet into solution (don’t vertex). Aliquot enough NAD Cycling
Enzyme mix (2 µl per assay) for the number of assays to be
performed in each experiment and freeze the stock solution
immediately at -80°C for future use. The enzymes are stable for
up to 2 months at -80°C after reconstitution.
b) Prepare a NAD Cycling Mix for each reaction:
NAD Cycling Buffer: 100 µl
NAD Cycling Enzyme Mix: 2 µl
c) Mix well and add 100 µl of the mix into each well of NADH
standard and samples.
4. Mix and incubate the plate at room temperature for 5 min to
convert NAD to NADH.
5. Add 10 µl NADH developer into each well. Let the reaction cycling
at room temperature for 1 to 4 hours or longer depend on the
reading of OD 450nm .
6. Read the plate at OD 450nm . The plate can be read multiple times
while the color is in developing. The reactions can be stopped by
adding 10 µl of Stop Solution into each well and mix well. The color
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should be stable within 48 hours in a sealed plate after addition of
Stop Solution.
5. Data Analysis
Apply the sample readings to NADH standard curve. The amount of
NADt or NADH in the sample wells can be calculated, then divide the
NADt or NADH amount by the sample amount (e.g. cell number or
extract protein amount) you added into the sample wells. The
concentration of NADt or NADH can be expressed in pmol/10 6 cells
or ng/mg protein (NADH molecular weight 664.4).
NAD/NADH Ratio is calculated as:
NADt – NADH
NADH
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6. Troubleshooting
Problem Reason Solution
Assay not
working
Unexpected
results
Assay buffer at
wrong temperature
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Measured at wrong
wavelength
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
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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Samples
with
inconsistent
readings
Lower/
Higher
readings in
samples
and
standards
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
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
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 Reason Solution
Standard
curve is not
linear
Not fully thawed kit
components
Pipetting errors when
setting up the
standard curve
Incorrect pipetting
when preparing the
reaction 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
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 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
For further technical questions please do not hesitate to
contact us by email (technical@abcam.com) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
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UK, EU and ROW
Email: technical@abcam.com
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: us.technical@abcam.com
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: hk.technical@abcam.com
Tel: 108008523689 ( 中 國 聯 通 )
www.abcam.cn
Japan
Email: technical@abcam.co.jp
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
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