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	final	unit definition	stock	final
	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)
Mohanty1997	100	10 to 100			1		18 nM
Komary2010		1			2.5

As a starting point for the development of our own application protocols (Krumschnabel 2015 Methods Mol Biol, MiPNet19.20) the OROBOROS-Team referred to two publications from Prof. Lazlo Tretter's O2k-Network Laboratory as a starting point for our development:

• Tretter 2012 Free Radic Biol Med
• Komary 2010 Biochim Biophys Acta.

Media

See Krumschnabel 2015 Methods Mol Biol.

Media with high antioxidant activity compete with HRP and partially consume H₂O₂ before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This was shown by comparing

• the sensitivity as determined by H₂O₂ additions to different media containing HRP and Amplex (R) UltraRed;
• the sensitivity as determined by addition of the actual fluorophore resorufin to the same media (signal change per added resorufin); the sensitivity is signal change per added H₂O₂ or resorufin.

The sensitivity, as determined by addition of H₂O₂, is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the sensitivity with respect to resorufin.

A chemical background drift, which is always observed for the Amplex /HRP system, is independent of the H₂O₂ sensitivity versus the same (absolute) drift expressed as (calibrated) "apparent H2O2 production" will be higher in a medium with low H2O2 sensitivity as compared to a medium with high H2O2 sensitivity.

As expected some traditional KCl based media, similar to those used in ... showed a far higher sensitivity against H2O2 addition than MiR05, while some experiments indicated a lower respiration.

Comparison MiR05Cr to MiRK03Cr

Instrumental settings

• Amp. Gain 1000
• Light intesity variable: 1 mA, 2 mA and 5 mA
• Configuration O2k-Fluo sensor always the same: O2k P1: Sensors B-0111/B-0082; O2k P2: Sensors B-0119/B-0117; O2k P3: Sensors A-0004/B-0112; O2k P4: Sensors A-0087/A-0088.

Media

• MiR05Cr
• MiRK03Cr: KCl 130 mM, HEPES free acid 20 mM, KH2PO4 10 mM, MgCl2 3 mM, EGTA 0.5 mM, BSA 0.1%, pH=7, add 120 mg Creatine to 40 ml buffer. After thawing MiRK03 a preciptate was observed, which was dissolved after 20 min shaking.

Titrations

• 10 µl Amplex UltraRed (stock: 1 mM -> 5 µM final in chamber)
• 4 µl HRP (stock: 500 U/ml -> 1 U/ml final in chamber)
• 2*10 µl of 56,32 µM H₂O₂ solution

Results/Figures

Drift vs Sensitivity:

• See Krumschnabel 2015 Methods Mol Biol.

Fig.6

Fig.7

--> Drift and sensitivity at least as good as MitoOx2 (=MiRK02)

Drift vs light intensity

Fig.14 and Fig. 15

--> MirK03, MiR05: drift not dependent on light intensity ( 1 to 5 mA)

compare to "MitoOx2":

Evaluation of respirometry in MiR05Cr versus MiRK03Cr (N=2):

Titrations

• 10 µl Amplex UltraRed (stock: 1 mM -> 5 µM final in chamber)
• 4 µl HRP (stock: 500 U/ml -> 1 U/ml final in chamber)
• 2*5 µl of 28.16 µM H₂O₂ solution : 0 µM, 0.07040 µM, 0.1406 µM

Fig.16 and 17

Fontanam 16:22, 6 August 2013 (CEST)

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