For research use only
rev. 05/11
ATP Colorimetric/Fluorometric Assay Kit
I.
(Catalog # GWB-AXR208; Store at -20°C)
Introduction:
ATP is the primary energy currency of living systems. Virtually all energy dependent
processes utilize the chemical energy stored in the phosphate bond of ATP. ATP is formed
exclusively in mitochondria and a variety of genetic diseases affect ATP formation in the
mitochondria. There are many commercially available ATP assays which detect femtomoles
or less of ATP using luminescence but these kits require specialized instrumentation and
utilize luciferase which can be difficult to maintain in active form. GenWay’s ATP Colorimetric
and Fluorometric Assay kit is designed to be a robust, simple method which utilizes the
Components
ATP Assay Buffer
ATP Probe (in DMSO)
ATP Converter
Developer Mix (lyophilized)
ATP Standard (1 µmol; lyophilized)
II.
II.
III.
IV.
GWBAXR208
Cap Code
Part Number
25 ml
0.2 ml
1 vial
1 vial
1 vial
WM
Red
Blue
Green
Yellow
GWB-AXR208 -1
GWB-AXR208 -2
GWB-AXR208 -3
GWB-AXR208 -4
GWB-AXR208 -5
phosphorylation of glycerol to generate a product that is easily quantified by colorimetric
(λmax = 570 nm) or fluorometric (Ex/Em = 535/587 nm) methods. The assay can detect
down to 50 pmol (1 µM) of ATP in various samples. The kit provides sufficient reagents for
100 assays.
Kit Contents:
Storage and Handling:
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.
Reagent Preparation:
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.
ATP Standard: Dissolve in 100 µl of distilled water to generate 10 mM stock solution. Keep
cold while using. Store at -20°C.
ATP Assay Protocol:
1. Standard Curve Preparations:
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 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.
For the fluorometric assay, further dilute the ATP Standard to 0.01- 0.1 mM with the dH2O
(Detection sensitivity is 10-100 fold higher with the fluorometric than with the colorimetric
assay). Follow the procedure as for the colorimetric assay.
2. Sample Preparation:
6
Tissue (1-10 mg) or cells (1 x 10 ) can be lysed in 100 µl of ATP Assay Buffer. 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 (GenWay, Cat. # GWB-AXR208). Centrifuge ice cold at
15,000xG for 2 minutes to pellet insoluble materials. 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.
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:
ATP Assay Buffer
ATP Probe
ATP Converter**
Developer Mix
Colorimetric Assay
44 µl
2 µl
2 µl
2 µl
Fluorometric Assay
45.8 µl
0.2 µl*
2 µl
2 µl
Mix well. Add 50 µl of the Reaction Mix to each well containing the ATP Standard and test
samples.
Notes: *For the fluorometric assay, use 1/10 of the probe to reduce fluorescence
background.
**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 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 O.D. 570 nm for colorimetric assay or Ex/Em = 535/587 nm for fluorometric assay
in a micro-plate reader. The signals are stable for over two hours.
6. Calculation: Correct background by subtracting the value derived from the 0 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:
C = Ts / Sv nmol/µl or µmol/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.
FOR RESEARCH USE ONLY! Not to be used on humans.
GenWay Biotech, Inc.
6777 Nancy Ridge Dr, San Diego, CA 92121 USA
Tel: 858-458-0866 | Fax: 858-458-0833
www.genwaybio.com | [email protected]
For research use only
rev. 05/11
GENERAL TROUBLESHOOTING GUIDE:
Problems
Cause
Solution
Assay not working
• Use of ice-cold assay buffer
• Assay buffer must be at room temperature
• Omission of a step in the protocol
• Refer and follow the data sheet precisely
• Plate read at incorrect wavelength
• Check the wavelength in the data sheet and the filter settings of the instrument
• Use of a different 96-well plate
• Fluorescence: Black plates ; Luminescence: White plates ; Colorimeters: Clear plates
• Use of an incompatible sample type
• Refer data sheet for details about incompatible samples
• Samples prepared in a different buffer
• Use the assay buffer provided in the kit or refer data sheet for instructions
• Samples were not deproteinized (if indicated in datasheet)
• Samples used after multiple free-thaw cycles
• Use the 10 kDa spin cut-off filter or PCA precipitation as indicated
• Use Dounce homogenizer (increase the number of strokes); observe for lysis under
microscope
• Aliquot and freeze samples if needed to use multiple times
• Presence of interfering substance in the sample
• Troubleshoot if needed, deproteinize samples
• Use of old or inappropriately stored samples
• Use fresh samples or store at correct temperatures till use
• Improperly thawed components
• Thaw all components completely and mix gently before use
• Use of expired kit or improperly stored reagents
• Always check the expiry date and store the components appropriately
• Allowing the reagents to sit for extended times on ice
• Always thaw and prepare fresh reaction mix before use
• Incorrect incubation times or temperatures
• Refer datasheet & verify correct incubation times and temperatures
• Incorrect volumes used
• Use calibrated pipettes and aliquot correctly
• Use of partially thawed components
• Thaw and resuspend all components before preparing the reaction mix
• Pipetting errors in the standard
• Avoid pipetting small volumes
• Pipetting errors in the reaction mix
• Prepare a master reaction mix whenever possible
• Air bubbles formed in well
• Pipette gently against the wall of the tubes
• Standard stock is at an incorrect concentration
• Always refer the dilutions in the data sheet
• Calculation errors
• Recheck calculations after referring the data sheet
• Substituting reagents from older kits/ lots
• Use fresh components from the same kit
• Measured at incorrect wavelength
• Check the equipment and the filter setting
• Samples contain interfering substances
• Troubleshoot if it interferes with the kit
• Use of incompatible sample type
• Refer data sheet to check if sample is compatible with the kit or optimization is needed
• Sample readings above/below the linear range
• Concentrate/ Dilute sample so as to be in the linear range
Samples with erratic readings
• Cell/ tissue samples were not completely homogenized
Lower/ Higher readings in Samples
and Standards
Readings do not follow a linear
pattern for Standard curve
Unanticipated results
Note: The most probable list of causes is under each problem section. Causes/ Solutions may overlap with other problems.
GenWay Biotech, Inc.
6777 Nancy Ridge Dr, San Diego, CA 92121 USA
Tel: 858-458-0866 | Fax: 858-458-0833
www.genwaybio.com | [email protected]