Talk:Amplex UltraRed

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MiPNet discussion forum: HRP independent background flux (2016-07-25)

Janne Purhonen

The problem was that I was observing a huge HRP independent sample related background. The assay was insensitive to addition of substrates and inhibitors. Then I came across a recent publication by Miwa et al. (Free Radical Bio Med 2016; 90:173 <> –183). They nicely showed that Amplex Red can cross the mitochondrial membranes, but HRP cannot, and the liver mitochondria express carboxylesterase which converts the Amplex Red to resorufin at high rate. The good thing is that this carboxylesterase can be inhibited for example by a commonly used serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) at 100 Β΅M which has marginal or no effect on mitochondrial respiration.
My own experimentations showed that 100 Β΅M PMSF reduced HRP independent sample related β€œH2O2” flux by 90% in the case of liver mitochondria and apparently completely eliminated this background in the case of kidney mitochondria. I also tested the PMSF in Amplex Red assay for isolated Drosophila melanogaster mitochondria in which the background is not that big issue as it is with the liver and kidney mitochondria. Anyway, PMSF eliminated the HRP-independent sample related background, thus inclusion of PMSF may improve the sensitivity of the assay not just with liver and kidney samples but with other sample types as well.
Purhonen J

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