Talk:Amplex UltraRed

Different Brands

Trade Mark	Manufacturer/ Distributor	price [€/mg]	product id
Amplex Red	Invitrogen	37.4	A12222
Amplex Ultra Red	Invitrogen	48.8	A36006
Ampliflu Red	Sigma	18.5	90101
Quanta Red	Thermo scientific		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 on thne other hand ctalase can be a valuebel tool for Talk:Amplex_red#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).