ab65349
NADP/NADPH Assay Kit
Instructions for Use
For the rapid, sensitive and accurate
measurement of NADP/NADPH in various
samples.
This product is for research use only and is not
intended for diagnostic use.
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Table of Contents
1.
Overview
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2.
Protocol Summary
3
3.
Components and Storage
4
4.
Assay Protocol
6
5.
Data Analysis
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6.
Troubleshooting
10
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1. Overview
Assays of nicotinamide nucleotides are of continual interest in the
studies of energy transforming and redox state of cells or tissue.
Abcam’s NADP/NADPH Assay Kit provides a convenient tool for
sensitive detection of the intracellular nucleotides: NADP, NADPH
and their ratio. The enzymes in the system specifically recognize
NADP/NADPH in an enzyme cycling reaction (It does not recognize
NAD+/NADH). There is no need to purify NADP/NADPH from sample
mix. The enzyme cycling reaction significantly increases detection
sensitivity. Results can be quantified using plate reader at OD450nm.
2. Protocol Summary
Sample Preparation
Standard Curve Preparation
Add NADP Cycling Mix
Measure Optical Density
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3. Components and Storage
A. Kit Components
Item
Quantity
NADP/NADPH Extraction Buffer
50 mL
NADP Cycling Buffer
15 mL
NADP Cycling Enzyme Mix
0.2 mL
NADPH Developer
Stop Solution
NADPH Standard (MW:833.36)
1 vial
1.2 mL
166.7 μg
Store kit at -80°C.

Ensure that the NADP Cycling Buffer is at room temperature
before use. The optimal temperature is 22°C. Keep other
enzymes on ice during the assay and protect from light as
much as possible.

Reconstitute NADPH standard with 200 μl pure DMSO to
generate 1 nmol/μl NADPH standard stock solution.
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
Reconstitute NADPH Developer with 1.2 ml of ddH2O.
Pipette up and down several times to completely dissolve
the pellet into solution.

Aliquot enough NADP 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 reconstituted enzymes are stable
for up to 2 months at -80°C.
B. Additional Materials Required

Microcentrifuge

Pipettes and pipette tips

Colorimetric microplate reader

96 well plate

Orbital shaker
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4. Assay Protocol
1. Sample Preparation:
a. For cell samples: Wash cells with cold PBS. Pellet 105 cells for
each assay in a microcentrifuge tube (2000 rpm for 5 min).
Extract the cells with 200 μl of NADP/NADPH 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 NADP/NADPH solution into a new labeled tube.
b. For tissue samples: Weigh ~20 mg tissue for each assay, wash
with cold PBS, homogenize with 400 μl of NADP/NADPH
Extraction Buffer in a microcentrifuge tube. Spin the sample at
14000 rpm for 5 min. Transfer the extracted NADP/NADPH
solution into a new labeled tube.
Note:
Cell or tissue lysates may contain enzymes that consume NADPH
rapidly. We suggest removal of these enzymes from the sample by
filtering the samples through 10 kDa molecular weight cut off filters
(ab93349) before performing the assays.
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2. Standard Curve Preparation:
Dilute 10 μl of the 1 nmol/μl NADPH standard with 990 μl
NADP/NADPH Extraction Buffer to generate 10 pmol/μl standard
NADPH (Note: diluted NADPH solution is unstable, must be used
within 4 hours).
Add 0, 2, 4, 6, 8, 10 μl of the diluted NADPH standard into labeled
96-well plate in duplicate to generate 0, 20, 40, 60, 80, 100 pmol/well
standard. Aliquot remaining standard and store at -20oC. Make the
final volume to 50 μl with NADP/NADPH extraction buffer.
a. To detect total NADP/NADPH: (NADPt):Transfer 50 μl of
extracted samples into labeled 96-well plate in duplicates.
b. To detect NADPH only: Aliquot 200 μl samples into eppendorf
tubes. Heat samples to 60°C for 30 min in a water bath or a
heating
block.
Under
the
conditions,
all
NADP
will
be
decomposed while NADPH will still be intact. Cool samples on
ice. Quick spin samples if precipitates occur.
Transfer 50 μl of NADPH samples into labeled 96-well plate in
duplicates
Several sample dilutions should be performed to ensure the reading
can be within the standard curve range.
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4. NADP Cycling Mix: Mix enough reagent for the number of
samples and standards to be performed. For each well, prepare a
total 100 μl NADP Cycling Mix:
NADP Cycling Buffer Mix:
NADP Cycling Enzyme Mix:
98 μl
2 μl
Mix well and add 100 μl Reaction Mix into each well; mix well.
5. Incubate the plate at room temperature for 5 min to convert NADP
to NADPH.
6. Development:
Add 10 μl NADPH Developer into each well. Let the reaction develop
for 1 to 4 hours. Read the plate at OD450nm.
Note: The signal increases with the reaction time. The plate can be
read multiple times while the color is developing. The reaction can
be stopped by addition of 10 μl Stop Solution each well and mix well.
The color should be stable within 48 hours in a sealed plate, after the
reactions are stopped.
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5. Data Analysis
Apply the sample OD450nm reading to standard curve. The
background reading is very significant in the enzyme cycling
reaction, therefore should be subtracted from standard and samples.
The amount of NADPt or NADPH can be expressed in pmol/106 cells
or ng/mg protein (NADPH molecular weight 745.4).
NADP/NADPH Ratio is calculated as:
NADPt - NADPH
NADPH
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6. Troubleshooting
Problem
Reason
Solution
Assay not
working
Assay buffer at
wrong temperature
Assay buffer must not be chilled
- needs to be at RT
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Unexpected
results
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
Measured at wrong
wavelength
Use appropriate reader and filter
settings described in datasheet
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
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
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
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
Wait for components to thaw
completely and gently mix prior
use
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
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 ([email protected]) 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: [email protected]
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: [email protected]
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: [email protected]
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: [email protected]
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
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All information / detail is correct at time of going to print.