Talk:Amplex UltraRed
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 | 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 cond)
| ||||
| Mohanty1997 | 100
|
10 to 100
|
1
|
18 nM
| |||
| Komary2010 | 1
|
2.5
|
|||||
At Oroboros Instruments we used two publications from Prof. Tretters OROBOROS MiPNet Reference Laboratory as a starting point for our development: Tretter 2012 Free Radic Biol Med and Komary 2010 Biochim Biophys Acta.
Media
Media that were designed to have some antioxidant properties will frequently consume H2O2 before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This can be 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)
(Sensitivity is signal change per added H2O2 or resorufin.)
While the sensitivity measured by the addition of H2O2 drops drastically from a simple phosphate buffer to media with strong antioxidant properties, this is not observed when the sensitivity is determined by the addition of resorufin.
Since the non biological drift always observed for the Amplex /HRP system is independent of the sensitivity versus H2O2 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 then MiR05, while some experiments indicated a lower respiration.
