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.
ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
Table of Contents
1.
Overview
3
2.
Protocol Summary
4
3.
Components and Storage
5
4.
Assay Protocol
7
5.
Data Analysis
9
6.
Troubleshooting
11
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
1. Overview
ATP is 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. There are ATP assays kits which
detects 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.
Abcam’s newly developed ATP Assay Kit (Colorimetric/Fluorometric)
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. The assay can detect as low as
50 picomol (1 µM) of ATP in various samples. The kit provides
sufficient reagents for 100 assays.
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
2. Protocol Summary
Sample Preparation
Standard Curve Preparation
Add Reaction Mix
Measure Optical Density or Fluorescence
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
3. Components and Storage
A. Kit Components
Item
Quantity
ATP Assay Buffer
25 mL
ATP Probe (in DMSO)
0.2 mL
ATP Converter
1 vial
Developer Mix (Lyophilized)
1 vial
ATP Standard (1 µmol; Lyophilized)
1 vial
* Store kit at -20°C, protect from light.
Warm ATP Assay Buffer to room temperature before use. Briefly
centrifuge all small vials prior to opening.
ATP PROBE: Ready to use as supplied. Warm to room temperature
prior to use to melt frozen DMSO. Store at -20°C, protect from light
and moisture. Use within two months.
ATP CONVERTER, DEVELOPER MIX: Dissolve in 220 µl ATP
Assay Buffer separately. Aliquot and store at -20°C. Use within two
months.
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
ATP STANDARD: Dissolve in 100 µl of distilled water to generate
10 mM stock solution. Keep cold while using. Store at -20°C.
B. Additional Materials Required
•
Microcentrifuge
•
Pipettes and pipette tips
•
Fluorescent or colorimetric microplate reader
•
96 well plate
•
Orbital shaker
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
4. Assay Protocol
1. Sample Preparation:
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a) Tissue (1-10 mg) or cells (1 x 10 ) can be lysed in 100 µl of ATP
Assay Buffer.
b) Due to the lability of ATP, for more accurate assays, the sample
should be quick frozen using liquid N2 or dry ice if it is to be
assayed at a later date. It is also recommended that the sample
be homogenized using perchloric acid. Alternatively, 10kDa spin
columns (ab93349) can be used to get rid of interfering enzymes
present in the sample.
c) Centrifuge ice cold at 15,000 x g for 2 minutes to pellet insoluble
materials.
d) Collect supernatant and add 2-50 µl to 96-well plate, bring final
volume to 50 µl/well with ATP Assay Buffer.
For unknown samples, we suggest testing several doses of your
sample to make sure the readings are within the standard curve
range.
2. Standard Curve Preparation:
a. For the colorimetric assay:
Dilute 10 µl of the ATP Standard with 90 µl of dH2O to generate
1 mM ATP standard, mix well. Add 0, 2, 4, 6, 8, 10 µl into a series
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
of wells and adjust volume to 50 µl/well with ATP Assay Buffer to
generate 0, 2, 4, 6, 8, 10 nmol/well of ATP Standard.
b. For the fluorometric assay:
Further dilute the ATP Standard to 0.01- 0.1 mM with dH2O
(Detection sensitivity is 10-100 fold higher with the fluorometric
than with the colorimetric assay). Follow the procedure as for the
colorimetric assay.
3. ATP Reaction Mix: Mix enough reagent for the number of
samples and standards to be performed: For each well, prepare a
total 50 µl Reaction Mix:
Colorimetric Assay
Fluorometric Assay
ATP Assay Buffer
44 µl
45.8 µl
ATP Probe
2 µl
0.2 µl*
ATP Converter**
2 µl
2.0 µl
Developer Mix
2 µl
2.0 µl
* Note: For the fluorometric assay, use 1/10 of the probe to reduce
fluorescence background.
**Note: Glycerol phosphate generates background. If significant
amount of glycerol phosphate is suspected in your sample, a
glycerol phosphate background control may be performed by
replacing the 2 µl ATP converter with 2 µl of ATP Assay Buffer. In
the absence of ATP converter, the assay detects only glycerol
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
phosphate, but not ATP. The glycerol phosphate background should
be subtracted from ATP reading.
4. Mix well. Incubate at room temperature for 30 minutes, protect
from light.
5.
Measure
OD
at
570
nm
for
colorimetric
assay
or
Ex/Em = 535/587 nm for fluorometric assay in a microplate reader.
The signals are stable for over two hours.
5. Data Analysis
Correct background by subtracting the value derived from the zero
ATP standard from all standard and sample readings.
Plot the standard curve. Apply ATP sample readings to the standard
curve. ATP concentration can then be calculated:
Concentration = Ts / Sv nmol/µl or mol/ml or mM
Where:
Ts is ATP amount in the reaction well from standard curve (nmol).
Sv is the sample volume added into sample wells (µl).
ATP molecular weight: 507.18 g/mol.
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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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ab83355 ATP Assay Kit (Colorimetric/Fluorometric)
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