ab176724
NADP/NADPH Assay Kit
(Fluorometric)
Instructions for Use
For monitoring NADP/NADPH activity in cell and
tissue extracts.
This product is for research use only and is not
intended for diagnostic use.
Version: 4 Last Updated: 18 March 2015
Table of Contents
1.
Overview
2
2.
Protocol Summary
5
3.
Kit Components
6
4.
Storage and Stability
7
5.
Materials Required, Not Supplied
7
6.
Assay Protocol
8
7.
Data Analysis
14
8.
Troubleshooting
16
1
1. Overview
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.
These methods suffer low sensitivity and high interference since the
assays are done in the UV range that requires expensive quartz
microplates.
The
low
sensitivity
of
the
absorption-based
NADP/NADPH assays makes the assays difficult to be automated
for high throughput screening that often uses small sample size.
2
Abcam's NADP/NADPH Assay Kit (Fluorometric) (ab176724)
provides a convenient method for sensitive detection of NADP,
NADPH and their ratio. The enzymes in the system specifically
recognize NADP/NADPH in an enzyme recycling 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 sample interference. 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
also provides NADP, NADPH extraction buffer, and cell lysis buffer.
3
Kit Key Features
 Broad
Application
–
can
be
used
for
quantifying
NADP/NADPH in solutions and in cell extracts
 Sensitive – detect as low as 10 picomoles of NADP/NADPH
in solution
 Continuous – suitable for both manual and automated
operations without a mixing or separation step
 Convenient – formulated to have minimal hands-on-time
 Non-radioactive – no special requirements for waste
treatment
4
2. Protocol Summary
Prepare NADPH standards and/or test samples
Add NADPH or NADP Extraction Solution
Incubate at RT for 15 min
Add NADP or NADPH Extraction Solution
Add NADP/NADPH reaction mixture
Incubate at RT for 15 min to 2 hours
Monitor fluorescence intensity at Ex/Em =540/590 nm
5
3. Kit Components
Item
Quantity
Storage
upon arrival
Storage after
use/
reconstitution
NADP/NADPH Recycling Enzyme
2 bottles
-20°C
-20°C
NADPH Sensor Buffer
20 mL
-20°C
-20°C
NADPH Standard (167 µg)
1 vial
-20°C
-20°C
NADPH Extraction Solution
10 mL
-20°C
-20°C
NADP Extraction Solution
10 mL
-20°C
-20°C
NADP/NADPH Control Solution
10 mL
-20°C
-20°C
NADP/NADPH Lysis Buffer
10 mL
-20°C
-20°C
Mixture (lyophilized powder)
6
4. Storage and Stability
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.
5. Materials Required, Not Supplied
 96 or 384-well solid black plate
 96 or 384-well white clear bottom plate
 Multi-well spectrophotometer (ELISA reader)
 Distilled water (ddH2O) or MilliQ
 PBS
7
6. Assay Protocol
1. Reagent Preparation
a)
NADPH Stock Solution:
Add 200 µL of PBS buffer into the vial of NADPH standard to
have 1 mM (1 nmol/µL) NADPH stock solution.
NOTE: The unused NADPH stock solution should be divided into
single use aliquots and stored at -20oC.
b)
NADP/NADPH reaction mixture:
Add 10 mL of NADPH Sensor Buffer to the bottle of
NADP/NADPH Recycling Enzyme Mixture and mix well.
NOTE: This NADP/NADPH reaction mixture is enough for two
96-well plates. The unused NADP/NADPH reaction mixture
should be divided into single use aliquots and stored at -20oC.
2. Sample preparation
a)
For lysis of plant cells:
Homogenize the leave 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.
8
b) For lysis of bacterial cells:
Collecting 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.
Centrifuge at
2500 rpm for 5 minutes, and then 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 10 µL of 1 mM NADPH stock solution (from Step 1a)
into 990 µL PBS buffer (pH 7.4) to generate 10 µM
(10 pmols/µL) NADPH standard solution.
NOTE: Diluted NADPH standard solution is unstable, and
should be used within 4 hours.
9
b)
Take 200 µL of 10 µM NADPH standard solution (from Step
3a) and perform 1:3 serial dilutions in PBS to get 3.3, 1.1,
0.37, 0.12, 0.04, 0.014 and 0 µM serially diluted NADPH
standards.
c)
Add
serially
diluted
NADPH
standards
and/or
NADP/NADPH containing test samples into a solid black
96-well microplate, as described in the tables below.
NOTE:
Prepare
cells
or
tissue
samples
as
desired.
NADP/NADPH Lysis Buffer can be used for lysing the cells for
convenience.
d)
For NADPH Extraction (NADPH): Add 25 μL of NADPH
Extraction Solution into the wells of NADP/NADPH
containing test samples. Incubate at room temperature for
10 to 15 minutes, then, add 25 μL of NADP Extraction
Solution to neutralize the NADPH extracts, as described in
the tables.
e)
For NADP Extraction (NADP): Add 25 μL of NADP
Extraction Solution into the wells of NADP/NADPH
containing test samples. Incubate at room temperature for
10 to 15 minutes, then, add 25 μL of NADPH Extraction
Solution to neutralize the NADP extracts, as described in
the tables.
f)
For Total NADP and NADPH: Add 25 μL of NADP/NADPH
Control Solution into the wells of NADPH standards and
NADP/NADPH containing test samples. Incubate at room
10
temperature for 10 to 15 minutes, and then add 25 μL of
Control Solution, as described in the tables.
NOTE:
Prepare
cells
or
tissue
samples
as
desired.
NADP/NADPH Lysis Buffer can be used for lysing the cells for
convenience.
BL
NS
1
NS
2
NS
3
NS
4
NS
5
NS
6
NS
7
NOTE:
BL
TS
TS
TS
(NADPH)
….
TS
(NADPH)
….
TS
(NADP)
….
TS
(NADP)
….
NS …. ….
1
NS
2
NS
3
NS
4
NS
5
NS
6
NS
7
NS = NADP/NADPH Standards, BL = Blank Control, TS =
Test Samples; TS (NADPH) = Test samples treated with NADPH
Extraction Solution for 10-15 minutes, then neutralized by NADP
Extraction solution; TS (NADP) = Test Samples treated with NADP
Extraction Solution for 10-15 minutes, then neutralized by NADPH
Extraction Solution.
11
Blank Control
Test
Sample
(NADP/NAD
PH)
Test
Sample
(NADPH
Extract)
Test
Sample
(NADP
Extract)
Serial
Dilutions*:
25 μL
PBS: 25 μL
Test
Sample:
25 μL
Test
Sample: 25
μL
Test
Sample: 25
μL
NADP/NAD
PH Control
Solution:
25 μL
NADP/NADPH
Control
Solution:
25 μL
NADP/NADP
H Control
Solution:
25 μL
NADPH
Extraction
Solution:
25 μL
NADP
Extraction
Solution:
25 μL
NADPH
Standard
Incubate at room temperature for 10 to 15 minutes
NADP/NAD
PH Control
Solution:
25 μL
NADP/NADPH
Control
Solution:
25 μL
NADP/NADP
H Control
Solution:
25 μL
NADP
Extraction
Solution:
25 μL
NADPH
Extraction
Solution:
25 μL
Total: 75 μL
Total: 75 μL
Total: 75 μL
Total: 75 μL
Total: 75 μL
*Note: Add the serially diluted NADPH standards from 0.01 μM to3
μM into wells from NS1 to NS7 in duplicate. High concentration of
NADPH (e.g., >100 μM, final concentration) may cause reduced
fluorescence signal due to the over oxidation of NADPH sensor (to a
non-fluorescent
product).
12
4. Run NADP/NADPH assay in supernatants reaction:
a)
Add 75 µL of NADPH reaction mixture (from Step 1b) into
each well of NADPH standard, blank control, and test
samples (from Step 3d) to make the total NADPH 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.
13
7. Data Analysis
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
NADPH reactions. The typical data are shown in Figure 1 (Total
NADP and NADPH vs. NADP or NADPH Extract).
14
Figure 1. Total NADPH and NAPD, and their extract dose response
were
measured
with
Abcam’s
NADP/NADPH
Assay
Kit
(Fluorometric) (ab176724) in a 96-well black plate using a microplate
reader. 25 µL of equal amount of NADP and NADPH was treated
with or without NADPH or NADP 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 NADPH 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).
15
8. 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: Solid black
plates;
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
16
Problem
Reason
Solution
Samples
with
inconsistent
readings
Unsuitable sample
type
Refer to datasheet for details
about incompatible samples
Use the assay buffer provided
(or refer to datasheet for
instructions)
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
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)
17
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
18
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19
All information / detail is correct at time of going to print.
Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.