Difference between revisions of "Talk:Amplex UltraRed"
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H<sub>2</sub>O<sub>2</sub> calibration with a '''56.32 µM''' H<sub>2</sub>O<sub>2</sub> solution | H<sub>2</sub>O<sub>2</sub> calibration with a '''56.32 µM''' H<sub>2</sub>O<sub>2</sub> solution | ||
''' | '''Commercial stock:''' Hydrogene peroxide solution, 3 wt. % (Sigma-Aldrich 323381-25ML) (Concentration = 880 mM H<sub>2</sub>O<sub>2</sub>) | ||
Dilution 1 (1:125): 200 µl commercial stock in 25 ml H<sub>2</sub>O = 7.04 mM H<sub>2</sub>O<sub>2</sub> solution | |||
Dilution 2 (1:125): 400 µl of dilution 1 in 50 ml H<sub>2</sub>O + 500 µl 1 mM HCl(for stabilization) = '''56.32 µM''' H<sub>2</sub>O<sub>2</sub> solution | |||
'''calibration for MiR05''': 2 * 10 µl in 2 ml chamber = 2* 0.2816 µM H<sub>2</sub>O<sub>2</sub> = total 0.5632 µM H<sub>2</sub>O<sub>2</sub> | '''calibration for MiR05''': 2 * 10 µl in 2 ml chamber = 2* 0.2816 µM H<sub>2</sub>O<sub>2</sub> = total 0.5632 µM H<sub>2</sub>O<sub>2</sub> | ||
See also: | |||
[http://www.oroboros.at/ | [http://www.oroboros.at/?O2k-Fluorescence Calibration template: Fluorescence_H2O2_calibration.xlsx] Further instructions are found there under 'Help'. | ||
Revision as of 08:14, 10 July 2013
Different Brands
| Trade Mark | Manufacturer/ Distributor | price [€/mg]
|
product id
|
| Amplex Red | 37.4
|
A12222
| |
| Amplex Ultra Red | 48.8
|
A36006
| |
| Ampliflu Red | 18.5
|
90101
| |
| Quanta Red | 15150
|
Conditions
| Citation | Amplex | HRP | pH | Limit of detection | |||
stock
|
work
|
unit definition
|
stock
|
work
|
|||
mM
|
µM
|
U/ml
|
U/ml
|
||||
| BIOTEK | 10
|
50
|
pyrogallol
|
10
|
0.1
|
7.4
|
4 nM (absorption 300 nm)
|
| INVITROGEN | 10
|
50
|
10
|
0.1
|
6 to 7.5
|
<80 nM
| |
| Towne 2004 | 160
|
0.41
|
7.5 to 8.5
|
100 nM
| |||
| Zhou1997 | 3 ?
|
0.3 to 1
|
50 nM (10 nm optimal cond)
| ||||
| Mohanty1997 | 100
|
10 to 100
|
1
|
18 nM
| |||
| Komary2010 | 1
|
2.5
|
|||||
At Oroboros Instruments we use the publication from Prof. Tretters OROBOROS MiPNet Reference Laboratory as a starting point for our development.
Substances incompatible with the Amplex Red method
The following substances/ classes of substances are strictly incompatible with the Amplex Red method for theoretical reasons:
- strongly redox active substances, e.g. cytochrome c, TMPD/Ascorbate
- inhibitors of horseradish peroxidase, see horseradish peroxidase
- catalase and other substances consuming H2O2. The effect of substances in the medium that consume H2O2 slowly is taken account of by the calibration procedure. However, such substances will decreases the sensitivity of the method. Note that catalase can be a value tool for Talk:Amplex_red#Checking_for_artifacts Checking for artifacts.
The effect of other substances should be checked by blank experiments, including comparing the sensitivity (result of calibration) before and after injecting the substances. In preliminary experiments Oroboros Instruments got indications that small amounts (as typically used in SUIT protocols) of the following substances are compatible with the method:
DMSO, ethanol, malate, glutamate, pyruvate, succinate, (ADP + Mg2+), (ATP + Mg2+), rotenone, FCCP, oligomycin, antimycin A, malonate, myxothiazol
Checking for artifacts
The Amplex method is based on the H2O2 dependent oxidation of Amplex Red to resorufin by HRP. Under unfavorable conditions Amplex Red may be oxidized even in the absence of H2O2. At a small rate such a oxidation occurs in the presence of HRP even without any sample present. The magnitude of this "Drift" depends inter alia on the used light intensity and can therefore be minimized by using the suggest or lower light intensity. Components of the sample may however induce a far higher, non H2O2 related rate of Ample Red oxidation. Therefore, especially when applying the method on new types of samples the method should be checked for artifacts. A few approaches are listed here:
Sequential addition of HRP and AmR: This method is particular easy to implement if Amplex Red and HRP have to be added to the chamber already containing the sample anyway: Inject first Amplex Red, wait a few minutes for flux stabilization. The Amp slope hast to stay near zero. Then add HRP. The Amp slope should increase and correspond to the H2O2 production. If a significant Amp slope is detected before the addition of HRP this increase in fluorescence is not caused by H2O2 production. The experiment can be continued as usual after this test. If the sample is injected routinely into the chamber already containing AmR and HRP the method can not be applied. In this case it is suggested to change this sequence at least for one experiment.
Addition of catalse: After a presumed H2O2 production rate is established a high dose (e.g. 10 µl of a 280000 u/ml stock solution) of catalase is injected. Because catalase competes with HRP for the available H2O2, the apparent H2O2 production rate (the Amp slope) has to be reduced nearly to zero. If the the Amp slope is unchanged or decreases only partially the increase in fluorescence is an artifact (non H2O2 dependent Amplex red oxidation). The experiment can NOT be continued afterwards!
Calibration with H2O2
H2O2 calibration with a 56.32 µM H2O2 solution
Commercial stock: Hydrogene peroxide solution, 3 wt. % (Sigma-Aldrich 323381-25ML) (Concentration = 880 mM H2O2)
Dilution 1 (1:125): 200 µl commercial stock in 25 ml H2O = 7.04 mM H2O2 solution
Dilution 2 (1:125): 400 µl of dilution 1 in 50 ml H2O + 500 µl 1 mM HCl(for stabilization) = 56.32 µM H2O2 solution
calibration for MiR05: 2 * 10 µl in 2 ml chamber = 2* 0.2816 µM H2O2 = total 0.5632 µM H2O2
See also: Calibration template: Fluorescence_H2O2_calibration.xlsx Further instructions are found there under 'Help'.
