ab83355
ATP Assay Kit
(Colorimetric/
Fluorometric)
Instructions for Use
For the rapid, sensitive and accurate
measurement of ATP in various samples.
This product is for research use only and is not
intended for diagnostic use.
Version: 8 Last Updated: 13 August 2014
1
Table of Contents
1.
Overview
3
2.
Protocol Summary
4
3.
Materials Supplied
5
4.
Storage and Stability
5
5.
Materials Required, Not Supplied
6
6.
Pre-Assay Preparation
7
7.
Assay Protocol
8
8.
Data Analysis
16
9.
Troubleshooting
20
10. FAQs
22
11. Quick Assay Procedure
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2
1. Overview
ATP (adenosine-5’-triphosphate) is a multifunctional nucleotide that
is most important in intracellular energy transfer, being the primary
energy currency of living systems. Virtually all energy requiring
processes utilize the chemical energy stored in the phosphate bond
of ATP. ATP is formed exclusively in the mitochondria and a variety
of genetic diseases can affect ATP formation in the mitochondria.
Abcam’s developed ATP Assay Kit (Colorimetric/ Fluorometric)
(ab83355) is designed to be a robust, simple method which utilizes
the phosphorylation of glycerol to generate a product that is easily
quantified by colorimetric (λ max = 570 nm) or fluorometric (Ex/Em =
535/587 nm) methods. There are other ATP assay kits which detect
femtomoles or less of ATP by measuring luminescence, but these
kits require specialized luminescence instrumentation and utilize
luciferase which can be difficult to maintain in active form.
ab83355 can detect as low as 1 µM of ATP in various samples. The
kit provides sufficient reagents for 100 assays.
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2. Protocol Summary
Standard Curve Preparation
Sample Preparation*
Add Reaction Mix & Incubate 30 min
Measure Optical Density or Fluorescence
*Samples may require deproteinization.
If you are an experienced user, please refer to Quick Assay
Procedure on page 23.
4
3. Materials Supplied
Item
Quantity
Storage
upon
arrival
Storage after
use/
reconstitution
ATP Assay Buffer
25 mL
-20°C
-20°C
ATP Probe (in DMSO)
200 μL
-20°C
-20°C
ATP Converter
1 vial
-20°C
-20°C
Developer Mix (Lyophilized)
1 vial
-20°C
-20°C
ATP Standard (1 µmol;
Lyophilized)
1 vial
-20°C
-20°C
4. Storage and Stability
Upon arrival, store kit at -20°C, protected from light. Kit can be
stored up to a year.
Ensure that ATP Assay Buffer is at room temperature before use.
Keep other enzymes on ice during the assay and protect from light.
Briefly centrifuge all small vials prior to opening.
Use reconstituted products within 2 months.
5
5. Materials Required, Not Supplied

MilliQ water or other type of double distilled water (ddH2O)

96 well plate: black plates (clear bottoms) for fluorometric
assay; clear plates for colorimetric assay

Microcentrifuge

Pipettes and pipette tips

Fluorescent or colorimetric microplate reader: switch on
before starting experiment

Heat block or water bath: switch on before starting
experiment

Vortex

Ice

Perchloric acid (PCA) 4M, ice cold (if performing
deproteinization step)

Potassium hydroxide (KOH), 2M (if performing
deproteinization step)

(optional): protease inhibitors
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6. Pre-Assay Preparation
1. ATP Standard:
Reconstitute ATP Standard (1 µmol) with 100µL of ddH2O to
generate a 10 mM ATP standard stock solution. Keep on ice
while in use. Aliquot ATP standard so that you have enough to
perform the desired number of assays (for example, 10 µL
aliquots). Store at -20°C.
2. ATP Probe:
Ready to use as supplied. Warm by placing in a 37°C bath for 1-5
min to thaw the DMSO solution before use. (Note: DMSO tends
to be a solid after -20°C storage, even when left at room
temperature so it needs to melt for a few minutes at 37°C).
Store at -20°C, protect from light. Once the probe is thawed, use
within two months.
3. ATP Converter:
Dissolve in 220 µL ATP Assay Buffer. Aliquot ATP converter
needed to perform the desired number of assays. Alternatively, a
good starting point would be 40 µL aliquots. Store at -20°C.
4. Developer Mix:
Dissolve in 220 µL ATP Assay Buffer. Aliquot developer needed
to perform the desired number of assays. Alternatively, a good
starting point would be 40 µL aliquots. Store at -20°C.
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Before you start the experiment:

Make sure all buffers and solutions are at room temperature,
and ATP standard is kept on ice at all times.

Make sure you have ice ready for your samples

Make sure the heat block/water bath and microplate reader are
switched on

Make sure you have the right plate type for your detection
method of choice at hand
7. Assay Protocol
1. Sample Preparation:
Different samples will contain different amount of ATP, so the
dilution range for each sample may vary; for instance, muscle
tissue can probably be diluted further than cultured cells as the
ATP concentration is generally higher.
The dilutions provided in this protocol are guidelines, so we
recommend that the first time you use a sample type, you
perform several different sample dilutions in the ATP Assay
Buffer to ensure readings fall within the standard curve range.
a) For cell (adherent or suspension) samples:
Harvest 106 cells for each assay.
i.
Wash cells with cold PBS.
ii.
Resuspend cells in 100 µl of ATP Assay Buffer. At this point
you can use protease inhibitors for your sample if
necessary.
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iii.
Homogenize cells quickly by pipetting up and down a few
times.
iv.
Centrifuge 2 min at 4°C (top speed using a microcentrifuge,
~13,000 rpm) to remove any insoluble material.
v.
Collect supernatant and transfer to a clean tube. Keep on
ice.
vi.
Perform deproteinization as described in section 7-step 1E.
b) Tissues:
Harvest samples: 1 – 10mg tissue for each assay.
i.
Wash tissue in cold PBS.
ii.
Resuspend tissue in 100 µl of ATP Assay Buffer. At this
point you can use protease inhibitors if necessary for your
sample.
iii.
Homogenize tissue with a Dounce homogenizer or pestle
sitting on ice, with 10 – 15 passes.
iv. Centrifuge
2–5
min
at
4°C
(top
speed
using
a
microcentrifuge, ~13,000 rpm) to remove any insoluble
material.
v.
Collect supernatant and transfer to a clean tube. Keep on
ice.
vi. Perform deproteinization as described in section 7-step 1E.
c) Plasma, serum and urine samples:
Plasma, serum and urine samples can be tested directly by
adding sample to the microplate wells.
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However, to find the optimal values and ensure your readings will
fall within the standard values, we recommend performing several
dilutions of the sample (1/2 – 1/5 – 1/10).
Maximum amount: 50 µl per well and per sample.
d) Red blood cells:
We recommend harvesting 107 RBCs as they are generally
smaller cells.
i.
Homogenize sample in 100 µl of ATP Assay Buffer.
ii.
Lyse cells by snap freeze-thaw cycles.
iii.
Centrifuge 2 min at 4°C (top speed using a microcentrifuge,
~13,000 rpm) to remove any insoluble material.
iv.
Collect supernatant and transfer to a clean tube. Keep on
ice.
v.
Perform deproteinization as described in section 7-step 1E.
e) Deproteinization step:
For biological samples with protein concentration < 20mg/ml
(tissue homogenate, cell lysates, urine):
i.
Take 550 µl of sample (if you have less amount of sample,
add ATP Assay Buffer), mix with 100 µl of ice cold PCA 4M
in 1.5 ml microcentrifuge tubes (you might need several),
vortex briefly and incubate on ice for 5 minutes.
ii.
Centrifuge at 13,000g for 2 min at 4°C and transfer
supernatant to a fresh tube. Measure volume of spnt.
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iii.
Precipitate excess PCA by adding an equal volume of icecold 2M KOH (to supernatant obtained in Step 1e-ii) and
vortexing briefly. After neutralization, It is very important
that pH = 6.8 – 7.2. Any left over PCA will interfere with the
assay.
iv. Centrifuge at 13,000g 15 min at 4°C and collect
supernatant.
For serum and other high concentration samples (> 20mg/ml):
i.
Take 400 µl of samples (if you have less amount of sample,
add ATP Assay Buffer), mix with 100 µl of ice cold PCA 4M
in 1.5ml microcentrifuge tubes (you might need several),
vortex briefly and incubate on ice for 5 minutes.
ii.
Centrifuge at 13,000g 2 min at 4°C and transfer
supernatant to a fresh tube. Measure volume of spnt.
iii.
Precipitate excess PCA by adding an equal volume of ice
cold 2M KOH (to supernatant obtained in Step 1e-ii) and
vortexing briefly. After neutralization, It is very important
that pH = 6.8 – 7.2. Any left over PCA will interfere with the
assay.
iv. Centrifuge at 13,000g 15 min at 4°C and collect
supernatant.
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2. Standard Curve Preparation:
a) For the colorimetric assay:
Dilute 10 μl of ATP standard 10 mM solution (see Pre-Assay
Preparation section) with 90 µl of ddH2O to generate 100 µl of
1mM ATP standard.
Prepare standard curve dilution as follows in a microplate or
microcentrifuge tubes:
END [ATP] IN WELL
ATP 1mM
standard
(µl)
Assay
Buffer (µl)
Total
Volume
0mM = 0 nmol/well
0 µl
150 µl
150 µl
0.04mM = 2 nmol/well
6 µl
144 µl
150 µl
0.08mM = 4 nmol/well
12 µl
138 µl
150 µl
0.12mM = 6 nmol/well
18 µl
132 µl
150 µl
0.16mM = 8 nmol/well
24 µl
126 µl
150 µl
0.2mM = 10 nmol/well
30 µl
120 µl
150 µl
Each dilution has enough amount of standard to set up 2
duplicates x 50 µl/well.
b) For the fluorometric assay:
Detection sensitivity is 10-100 fold higher for a fluorometric than a
colorimetric assay, so standard needs to be diluted accordingly.
12
Prepare a 1mM ATP standard as described in step 2b.
Dilute 10 µl of 1mM ATP standard onto 90 µl to create 100 µl of
0.1 mM standard.
Prepare standard curve dilution as follows in a microplate or
microcentrifuge tubes:
END [ATP] IN WELL
ATP 0.1mM
standard
(µl)
Assay
Buffer (µl)
Total
Volume
0 µM = 0 nmol/well
0 µl
150 µl
150 µl
4 µM = 0.2 nmol/well
6 µl
144 µl
150 µl
8 µM = 0.4 nmol/well
12 µl
138 µl
150 µl
12 µM = 0.6 nmol/well
18 µl
132 µl
150 µl
16µM = 0.8 nmol/well
24 µl
126 µl
150 µl
20µM = 0.10 nmol/well
30 µl
120 µl
150 µl
Each dilution has enough amount of standard to set up 2
duplicates x 50 µl/well.
NOTE: If your sample readings fall out the range of your fluorometric
standard curve, you might need to adjust the dilutions and create a
new standard curve (see FAQs section for more information).
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3. ATP Reaction Mix:
a) Prepare ATP Reaction Mix for each reaction:
Colorimetric
Fluorometric
44 µl
45.8 µl
ATP Probe*
2 µl
0.2 µl
ATP Converter
2 µl
2 µl
Developer Mix
2 µl
2 µl
50 µl
50 µl
ATP Assay Buffer
*For fluorometric reading, use 0.2μl/well of the Probe to decrease
the
background
readings,
therefore
increasing
detection
sensitivity.
NOTE: Prepare a Master Mix to ensure consistency. We
recommend that for each of the components you add Number of
samples + 1. For example, to measure standards and 2 samples
in duplicate (a total of 16 samples), you will prepare the following
master mix for 17 samples:
Colorimetric
Fluorometric
748 µl
778.6 µl
ATP Probe*
34 µl
3.4 µl
ATP Converter
34 µl
34 µl
Developer Mix
34 µl
34 µl
850 µl
850 µl
ATP Assay Buffer
Master mix for 17
NOTE: Glycerol phosphate generates background. If you suspect
there is a significant amount of glycerol phosphate in the sample,
14
perform a glycerol phosphate control by omitting the ATP converter
in your reaction mix as shown below.
Colorimetric
Fluorometric
46 µl
47.8 µl
ATP Probe
2 µl
0.2 µl
Developer Mix
2 µl
2 µl
50 µl
50 µl
ATP Assay Buffer
In absence of ATP converter, the assay detects only glycerol
phosphate but not ATP. The glycerol phosphate background should
then be subtracted from your sample ATP reading.
4. Standard and sample plate set-up
There is no specific pattern for using the wells on the plate for the
reading. However, a standard curve in duplicate has to be
assayed with the samples, as well as background wells.
We suggest that each sample is assayed at least in duplicate. As
mentioned previously, we recommend performing different
sample dilutions in ATP Assay Buffer to ensure readings fall
within the standard curve range.
Prepare the plate as follows:
a) Add 50 µl of ATP reaction mix (with or without ATP converter) to
each well.
b) Add 50 µl of standard or sample to each well as described in Step
2.
c) Incubate at room temperature for 30 min protected from light.
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d) Measure output on a microplate reader.
Colorimetric assay: measure OD570nm.
Fluorometric assay: measure Ex/Em = 535/587nm
The reaction is stable for at least 2 hours.
8. Data Analysis
Calculation:
a) Determine the average absorbance of each duplicate standard.
b) Subtract the absorbance value of the blank from itself and all
other standards and samples. This is the corrected reading.
Note: If samples contain glycerol phosphate, you should have
done a sample background control by omitting the ATP
converter to determine glycerol phosphate background (Step3b
previous section).
NOTE: Correct background by subtracting the value derived from
the zero ATP standard from all readings. Plot the standard curve.
Apply sample readings to the standard curve.
c) Plot the corrected reading values (from Data Analysis Step b) of
each standard as a function of the amount of ATP. See figures 1
and 2 for typical standard curves.
d) Calculate the trendline equation based on your standard curve
data.
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e) Calculate Ts (ATP amount from standard curve) using the
corrected reading of each sample and the equation:
𝑇𝑠 = 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 ‒ (𝑦 ‒ 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)
𝑆𝑙𝑜𝑝𝑒
(
)
f) ATP concentration can then be calculated:
[ATP] (nmol/µl or mM) = Ts / Sv
Where:
Ts is ATP amount from standard curve (nmol or mM).
Sv is the sample volume (before dilution) added in sample wells (μl).
Figure 1: Typical ATP standard calibration curve using colorimetric
reading.
17
Figure 2: Typical ATP standard calibration curve using fluorometric
reading.
To calculate the ATP concentration in mg ATP/g:
The molecular weight of ATP is 507.18 g/mol; therefore, the
concentration of 1mM ATP standard = 0.507 µg//µl (colorimetric
standard curve) and of 0.1mM ATP standard = 50.7 ng/µl
(fluorometric standard curve).
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Figure 3: Quantitation of ATP in fish liver (2.5 µl of 10 times diluted
sample), fish muscle (5 µl of 10 times diluted sample) and Jurkat cell
lysate (5 µl) using fluorometric assay. Samples were spiked with
known amounts of ATP (300 pmol).
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9. 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
Samples
with
inconsistent
readings
Re-read and follow the protocol
exactly
Ensure you are using
appropriate reader and filter
settings (refer to datasheet)
Fluorescence: Black plates
(clear bottoms);
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
Unsuitable sample
type
Sample readings are
outside linear range
Use recommended sample
types as listed on the datasheet
Concentrate/ dilute samples to
be in linear range
Unsuitable sample
type
Cell/ tissue samples
not sufficiently
homogenized
Too many freezethaw cycles
Samples contain
impeding substances
Refer to datasheet for details
about incompatible samples
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
Samples are too old
or incorrectly stored
20
Problem
Lower/
Higher
readings in
samples
and
standards
Standard
curve is not
linear
Reason
Not fully thawed kit
components
Out-of-date kit or
incorrectly stored
reagents
Reagents sitting for
extended periods on
ice
Solution
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
Incorrect incubation
time/ temperature
Refer to datasheet for
recommended incubation time
and/ or temperature
Incorrect amounts
used
Check pipette is calibrated
correctly
Not fully thawed kit
components
Pipetting errors when
setting up the 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
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10.
FAQs
1. Which type of ATP is detected with this kit?
This kit detects total amount of active ATP in the cell, including
both cellular and mitochondrial.
It does not detect the nucleotide dATP (deoxyadenosine
triphosphate).
2. My samples (cells/tissue) have been frozen prior to starting
the experiment. Can I still use them?
Due to the lability of ATP, we strongly recommend that you use
fresh samples. However, if you cannot perform the assay at the
same time, we suggest that you snap freeze cells or tissue in
liquid nitrogen or dry ice upon extraction and store the samples
immediately at -80°C. When you are ready to prepare your
samples for the assay, thaw them on ice. Be aware however that
this may affect the stability of your samples and the readings can
be lower than expected.
3. My sample reading seems to fall below the range of the
fluorometric standard curve.
As previously mentioned, fluorometric detection is about 10 – 100
times more sensitive than colorimetric. In this protocol we only
show how to prepare a standard curve that covers a range of
10µM – 0.1mM ATP. If your samples fall below, you could
prepare a 0.01mM ATP standard (by diluting 0.1mM ATP 10
times further) and prepare a standard curve following the same
procedure as described in section 2b. Remember that the
readings you obtain will be 10 times more diluted and therefore
you will have to adjust your calculation.
4. When used colorimetrically, the assay develops a red color.
I’m using cell culture media as sample, which contains a dye
that makes it look red. Will this interfere with the absorbance
readings?
22
The color red comes from the reflection of red light and
absorption of other wavelengths of light, including 570nm. The
reflection and absorption are both proportional to the amount of
the colored product in the well: the redder the color looks in the
well, the higher the absorbance reading should be. Colored
substances in the sample (such as the dye found in the majority
of culture media) can sometimes contribute to background.
We recommend a control well with just media or sample buffer +
ATP reaction mix to assess the background.
5. Can I use Abcam’s 10kDa spin columns (ab93349) for
deproteinizing the samples instead of doing the
deproteinization step you recommend?
Both 10kD spin columns and PCA precipitation protocol are valid
methods for deproteinizing the samples.
The 10kD spin column is a quicker method as it only requires a
quick spin in the microcentrifuge. However, it can be difficult to
obtain a good eluate if the sample contains a large amount of
protein or there is some precipitate or particulates in the solution.
The perchloric acid (PCA) precipitation protocol is a bit more
laborious and requires access to perchloric acid, but it is the most
efficient method to get rid of unwanted proteins and enzymes,
and it is the recommend method if you have a large amount of
protein in your sample.
23
11.
Quick Assay Procedure
NOTE: This procedure is provided as a quick reference for
experienced users. Follow the detailed procedure when initially
performing the assay.
1. Solubilize ATP standard, thaw ATP probe and prepare enzyme
mix (aliquot if necessary); get equipment ready
2. Prepare appropriate standard curve for your detection method of
choice (see page 12 – colorimetric, page 13 – fluorometric)
3. Prepare samples in duplicate (find optimal dilutions to fit standard
curve readings)
4. Prepare ATP Reaction Mix (Nb samples + Standards + 1)
Colorimetric
ATP Assay Buffer
Fluorometric
44 µl
45.8 µl
ATP Probe
2 µl
0.2 µl
ATP Converter
2 µl
2 µl
Developer Mix
2 µl
2 µl
50 µl
50 µl
5. Set up plate for standard and samples
50 µl standard/sample
50 µl ATP Reaction Mix
6. Incubate plate at RT 30min
7. Measure plate at OD570nm for colorimetric assay or Ex/Em =
535/590 nm for fluorometric assay.
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Email: [email protected] | Tel: 030-896-779-154
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Email: [email protected] | Tel: 911-146-554
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Email: [email protected]
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
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Email: [email protected] | Tel: 888-77-ABCAM (22226)
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Japan
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All information / detail is correct at time of going to print.
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