Active motif europium tetracycline (eutc) peroxide assay manual

Europium Tetracycline (EuTc)
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:
Information in this manual is subject to change without notice and does not constitute a commit-ment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit or implied. Information may be changed or updated in this manual at any time.
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The manufacturer of this documentation is Active Motif, Inc.
2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective companies.
TABLE OF CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Assay Principle and Performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Components and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Protocols
A. Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 B. Cuvette Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 C. 96-well Microplate Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Section B. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Introduction
Hydrogen peroxide (H O ) is a reactive metabolic by-product that is a key regulator in a number of oxidative stress-related states. Functioning through NFkB and other factors, hydroperoxide- mediated pathways have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteo-porosis, a number of neurodegenerative diseases and Down’s syndrome. Due to its implication in these many disease states, there is much interest in sensitive assays to monitor hydrogen peroxide in biochemical, clinical, as well as in environmental samples. In addition, there is much research on the enzymes that produce and eliminate hydrogen peroxide, and on their inhibitors.
Active Motif’s EuTc-Hydrogen Peroxide Assay is based on a novel fluorescent reagent that is used to detect free or enzymatical y produced H O . Europium Tetracycline (EuTc) detects hydrogen peroxide in a fast and easy method, and without the need for additional enhancer solutions. This makes EuTc a sensitive reagent to monitor catalases, peroxidases or glucose-oxidase activities through in vitro enzymatic assays, or to study substances that inhibit or activate these enzymes. The main advantages of using EuTc for luminescent peroxide detection are: • Fast, 10-minute incubation time• Applicable to turbid samples• Functions at neutral pH• No need for enzymatic amplification• Large Stoke’s shift decreases interference of background fluorescence• Long decay time enables time-resolved, or time-gated, measurement catalog no.
*Sufficient EuTc is provided for 1000 rxns performed in 96-well plates, or 100 rxns in cuvettes.
The EuTc-H O Assay is for research use only. Not for use in diagnostic procedures.
Assay Principles and Performance
Europium tetracycline (EuTc) alone is weakly fluorescent. But, it combines rapidly with free H O to form a EuTc-H O complex that is 15-fold brighter than EuTc alone (Figure 1) Thus, the basic principle of EuTc assays is based upon the following equilibrium: Figure 1: Absorption and emission spectra of EuTc before and after addition of hydrogen peroxide.
In addition to measuring H O levels via the increase in fluorescent intensity, EuTc makes it pos- sible to measure the change in decay time (lifetime) of the EuTc-H O complex (Table 1). The use of lifetime-based measurement has significant advantages when measuring hydrogen peroxide levels in biological fluids as such samples typical y have high levels of auto-fluorescence that can interfere with intensity-based measurements. Because the EuTc-H O signal is relatively long lived, lifetime measurement can completely eliminate any background contributed by the light source, the biological sample (typical y ~0.1-10 ns) or by free EuTc.
Dye Complex
Absorption (nm)
Emission (nm)
Time (µs)
Table 1: Lifetime measurements of free and peroxide-bound EuTc.
Assay time: 30 minutes.
Detection limit: 0.96 µM of H O .
Interferences: Phosphate and citrate cause an increase in fluorescence intensity of EuTc and may
interfere with the increase in fluorescence caused by EuTc- H O -complex formation. Detergents
should also be avoided as they can interfere with the EuTc reagent.
Components and Storage
EuTc is supplied as a dry powder that can be stored at room temperature. After resuspension, it
should be stored at -4°C in the dark.
Reagents
Quantity
Storage / Stability
Additional materials required for both 96-well plate and cuvette assays
Additional materials required for 96-well plate assays only
• 6 x 1 ml reaction tubes• Reaction tubes, 2 ml minimum• Fluorescence microplate reader, excitation of 395-405 nm / emission of 617 nm Additional materials required for cuvette assays only
• 6 x 10 ml glass flasks• Reaction tubes, 2 ml minimum• Cuvettes• Fluorescence reader, excitation of 395-405 nm / emission of 617 nm Protocols
A. Buffer Preparation and Recommendations
Preparation of Solution A (EuTc Working Solution)
• Dissolve the entire amount of the supplied EuTc in 100 ml of distilled water.
• Its absorbance at 405 nm is ~0.76 per cm.
• Store the EuTc Working Solution at 4°C in the dark.
• EuTc Working Solution is stable for one month if kept in the dark.
Preparation of H O Stock Solution
• Add 810 µl of 30% hydrogen peroxide into a final volume of 10 ml distilled water.
• H O Stock Solution is stable for one month when kept at 4ºC Preparation of Solution B (400 µM hydrogen peroxide standard)
• Add 50 µl of H O Stock Solution (prepared above) into a final volume of 100 ml distilled H O.
• Solution B should be prepared freshly for each new experiment. B. Cuvette Assay
Preparation of the Calibration Graph
1.
Make 6 dilutions of Solution B in distilled water in 10 ml flasks at room temperature: (400 µM) (320 µM)
(200 µM)
Total Volume
2. Add 1 ml each of the diluted solutions to a 2 ml reaction tube. Work at least in duplicates.
3. Add 1 ml of Solution A to each tube, mix by pipetting up and down, then incubate for 4. In a separate tube, mix 1 ml of distilled water with 1 ml of Solution A for use as a blank.
5. Pipette each reaction mix into a cuvette and measure the luminescence intensity against the blank. Choose an excitation wavelength of 395-405 nm and an emission wavelength of 617 nm (at 8-10 nm bandwidth).
6. Calculate the average value for the duplicate measurements, then plot the fluorescence intensity (or relative increase in fluorescence intensity) versus the concentration of H O .
Determination of H O Concentrations in Samples
Mix 1 ml of aqueous sample containing 0.1-10 mg/ml of H O with 1 ml of Solution A in a 2 ml reaction tube and incubate for 10 minutes at room temperature. As phosphate buffer can cause high background, we recommend samples in phosphate-free buffers like MOPs.
2. Pipette each solution into a cuvette and measure the luminescence as described above.
3. Use the Calibration Graph prepared above and the measured luminescence to determine the concentration of H O for each sample.
C. 96-well Microplate Assay
Preparation of the Calibration Graph
1.
Make 6 dilutions of Solution B in distilled water in 1 ml tubes at room temperature: (400 µM) (320 µM)
(200 µM)
Total Volume
2. Pipette 100 µl each of the diluted solutions into a well of a 96-well microplate. Work at least 3. Add 100 µl of Solution A to each tube, mix and incubate for 10 minutes at room temperature.
4. In a separate wells, mix 100 µl of distilled water with 100 µl of Solution A for use as a blank.
5. Read the plate on a fluorescence microplate reader set to an excitation wavelength of 395-405 nm and an emission wavelength of 617 nm. If a time-resolving reader is available, set the lag time to >30 µs and integrate over 100 µs.
6. Calculate the average value for the duplicate measurements, then plot the fluorescence intensity (or relative increase in fluorescence intensity) versus the concentration of H O .
Determination of H O Concentrations in Samples
Mix 100 µl of aqueous sample containing 0.2-10 mg/ml of H O with 100 µl of Solution A in a well of a 96-well microplate and incubate for 10 minutes at room temperature. As phosphate buffer can cause high background, we recommend samples in phosphate-free buffers like MOPs.
2. Measure the luminescence as described above.
3. Use the Calibration Graph prepared above and the measured luminescence to determine the concentration of H O for each sample.
Appendix
Section A. Troubleshooting Guide
Problem/question
Recommendation
Phosphate and citrate can cause increases in the fluorescent intensity of the EuTc-H O complex. Use phosphate-free buffer systems like MOPs buffer.
Set the lag time of the reader above 30 µs.
It is important to prepare the Standard Curve in the same media as that of the sample. Use equal volumes of buffer in the sample and in the dilutions when preparing the Standard Curve.
Section B. Related Products
MAX Stain™ Immunofluorescence Tools
Catalog No.
(contains 1 each of 15252, 15253 & 15254) Cell Viability Assay
Catalog No.
Transcription Factor ELISAs
Catalog No.
Fluorescent Cell Stains
Catalog No.
Chromeo™ Live Cell Mitochondrial Staining Kit Chromeo™ Red Fluorescent Fixed Cell Staining Kit Protein & Extract Quantification
Catalog No.
Capillary Electrophoresis
Catalog No.
Fluorescent Pyrylium Dyes (Py-Dyes)
Catalog No.
Fluorescent Dyes
Excitation / Emission
Catalog No.
Antibody/Protein Labeling
Excitation / Emission
Catalog No.
Fluorescent Secondary Antibodies
Catalog No.
Fluorescent Protein Labeling
Catalog No.
Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
Germany Free Phone: 0800 181 99 10 Telephone: Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:
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