Hey
can anyone recommend a good way to stop/quench H2O2 when treating plant tissues? The tissue should survive.
Best and thanks a lot for your help
Sebastian
Hi Sebastian,
Long time no see! I'm currently writing a manuscript that included various techniques to visualize and block/scavenge ROS and H2O2. In Arabidopsis, for us Potassium Iodide (KI) worked really well to remove H2O2 accumulation and diphenyleneiodonium (DPI) worked well to reduce NADPH oxidase-mediated H2O2 production. For methods and some results, see chapter 4 (p89 & p96) of the thesis of my colleague Zeguang Liu: https://dspace.library.uu.nl/handle/1874/378724 .
Send me a message if I can be of more help.
Edit: DPI has clear side-effects and should be used with caution (see other comments of Markus and Ian).
Good luck!
Hi Sebastian,
Long time no see! I'm currently writing a manuscript that included various techniques to visualize and block/scavenge ROS and H2O2. In Arabidopsis, for us Potassium Iodide (KI) worked really well to remove H2O2 accumulation and diphenyleneiodonium (DPI) worked well to reduce NADPH oxidase-mediated H2O2 production. For methods and some results, see chapter 4 (p89 & p96) of the thesis of my colleague Zeguang Liu: https://dspace.library.uu.nl/handle/1874/378724 .
Send me a message if I can be of more help.
Edit: DPI has clear side-effects and should be used with caution (see other comments of Markus and Ian).
Good luck!
Hey Sjon
nice to hear from you :-) yes long time no see. Thanks a lot for the advise I will have a look on it.
Best wishes
Sebastian
Please look at the following below links which may help you in your analysis:
http://www.ihcworld.com/_technical_tips/peroxidase_tips.htm
https://www.scielo.br/pdf/txpp/v25n4/03.pdf
Thanks
Hi Sebastian
Markus is entirely right in recommending that DPI should not be used as an inhibitor on intact plant tissues, as there are far too many effects. In addition to the effects he mentions, DPI also inhibits the mitochondrial NAD(P)H dehydrogenases (including Complex I) some with very low Ki (see Agius et al. 1998 Physiologia Plantarum 104, 329-336, Roberts et al. 1995 FEBS Letters 373, 307-309).
Ascorbate does sound like a good possibility.
Best wishes
Max
Dear colleagues,
I performed now a treatment of my plants with different concentrations of H2O2 in liquid with different concentrations of H2O2 (2mM and less). I treated for 15 min and quenched (or wanted to do so) with equimolar concentrations of ascorbic acid. as a control I added the highest used concentration of ascorbic acid to plant material without prior treatment of H2O2. Unfortunately all plants except this untreated control died after transfer on agar plates.
Do you think I should add HRP to the quenching reaction? Or should I keep plates in the dark for a certain time after the treatment?
Best and thanks a lot for your support!
Dear Sebastian
If you only want the treatment to last for 15 min, you will have to remove the H2O2 from the solution at the end of the incubation. There are two ways you can do that: (i) remove the plants from the solution, rinse them with the next medium (containing ascorbate) and place them in that solution for a suitable amount of time before plating them. (ii) add catalase to the medium with the ascorbate to remove the H2O2.
Good luck
Max
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