• Katarina Venglovska added an answer:
    Does anyone have any suggestions on doing an SOD assay using the Marklund and Marklund protocol?
    I am performing the SOD assay using the pyrogallol protocol by Marklund and Marklund for estimating SOD from serum. I am not getting the desired results. I'm using a 30 mM concentration of pyrogallol, but my reading of serum is more than the pyrogallol reading.
    Can anyone suggest any solutions.
    Katarina Venglovska · Slovak Academy of Sciences

    From my point of view, the activity of SOD in serum is very low and it is under limit of detection, that's why your reading is more than pyrogallol reading. I am using this method for SOD measurement in various tissues, but I have never tried to measure the activity in serum. But if I have similar results as you, I prepare more concentrated solution and if I don't have the difference between readings I dilute the sample. I always calculate the % of inhibition during measurement, as % Inhibition = [1-(dA/min of sample / dA/min of blank)] x 100%. And I measure the absorbance in time intervals at 420 for 4 minutes with one minute delay because from my experience is the reaction in first minute unstable.

  • Shane Vontelin van Breda added an answer:
    Can anyone give advice on Fenton reaction inhibition in M. tuberculosis?
    I am investigating the mechanism of action of a cationic peptide against MTB. I suspect one of the mechanisms is by the Fenton reaction. One way I would like to prove this is by use of thiourea and dipyridyl, as they have been used before in inhibiting the Fenton reaction.

    Does anybody have an idea of what concentrations I should begin looking at against MTB?

    Has anybody got any experience investigating and inhibiting Fenton reaction again against MTB?

    I would also like to ask about the preparation of thiourea and dipyridyl. Should it be added to bacterial media, then autoclaved? Or should the compounds be added to the autoclaved media using filter sterilization?

    Shane Vontelin van Breda · University of Pretoria

    Thank you Boris.

  • Alexandra De Sousa asked a question:
    Why should blank samples not have PMS in scavenging activity of anion superoxide?
    I´m following this protocol:

    Superoxide radicals are generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of NBT. In these experiments, the superoxide radicals were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 mM) solution, 1 ml NADH (78 mM) solution and sample solution of fraction (25 – 500 mg/ml) in ethanol. The reaction started by adding 1 ml of PMS solution (10 mM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min, the absorbance was read at 560 nm by spectrophotometer (Schimadzu UV-Vis 1700) against blank samples using ascorbic acid as a control. Decreased absorbance of the reaction mixture indicated the increasing of superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:
    % inhibition = [(A0-A1)/A0] x 100
    where A0 was the absorbance of the control , and A1 was the absorbance in the presence of fraction or standards.
  • Just Genius added an answer:
    Has anyone detected superoxide anions with luminescence method?
    For measurement of superoxide anions induced by Angiotensin II in VSMC cell lines, I used luminol and enhancer incubated with pretreated cells but the value of light intensity was only about 200 per 105 cells after 30 mins incubation. Could that be right? Why is the light intensity so low?
    Just Genius · University Hospital Essen
    Hi again! As far as I know, luminol will primarily detect hydrogen peroxide, not superoxide. Lucigenin may be the better probe, but you may also consider fluorescent dyes (e.g. available from Molecular Probes). In the experimental setup you describe, light emission will be dependent on the presence of (intrinsic) SOD which converts superoxide into H2O2. The readings of your instrument are arbitrary and depend on the detector setup and the instrument settings (integration time, blank value substraction detector gain etc), so 200 counts may be entirely OK. You may use xanthine+xanthineoxidase as a positive control (it will generate superoxide anions). Or you treat some leucocytes with LPS and measure the oxidative burst (works even with full blood). This would be a nice "biological" positive control.
    Good luck!
  • Praveen Roylawar asked a question:
    What si the best protocol for NBT staining in tomato leaves for detection of superoxide radicals?
    Can anyone please suggest the best protocol for NBT staining (for suproxide radicals) in tomato leaves. Could you also suggest how, after staining, I can efficiently remove chlorophyll without damaging leaves and heat burning.
  • Christian Q. Scheckhuber added an answer:
    What is best chemical to induce oxidative stress selectively by superoxide anions?
    But not by other ions such as hydroxyl etc. I wants to elevate superoxide level selectively in the cells.
    Christian Q. Scheckhuber · Senckenberg Research Institute
    You could think about using Paraquat (also known as methyl viologen). It is supposed to generate superoxide anions via complex I of the mitochondrial respiratory chain. It is commercially available.
    I hope this information helps you.

    Best regards
  • Olivier Coste asked a question:
    Can antioxidant molecules interfere with an NBT assay for oxidative burst and give a "false negative"?
    If my molecule activates macrohpages oxidative burst but at the same time scavenges the superoxide radicals, is it going to give a false negative?
  • Reena Arora asked a question:
    Does anyone have experience with MitoSOX assay?
    I was wondering if anyone has done a MitoSOX assay in 96 well plate?
  • Devendra Kumar added an answer:
    When is the exact time that superoxide radicals are produce in plants?
    In a walnut.
    Devendra Kumar · Indian Agricultural Research Institute
    its also depend on type of pathogen, resistance gene...but most case within 30 minute........reference-NO way back: nitric oxide and programmed cell death in
    Arabidopsis thalianasuspension cultures
    Andrew Clarke, Radhika Desikan, Roger D. Hurst, John T. Hancock and Steven J. Neill*
  • Maan Hayyan added an answer:
    What is superoxide exactly?
    Superoxide radical (O2.- ) and Superoxide anion(O2- ) appear in most of literature. From the structure, superoxide is formed after oxygen received one electron. I think, superoxide means "superoxide anion". What do you think?
    Maan Hayyan · University of Malaya
    Superoxide ion is an anionic radical and it behaves either as an electron donor, an electron reducing agent, an oxidant, a base or as a nucleophile.
    Superoxide ion-radical has been known since 1934, when Haber and Weiss have proposed that O2- can be formed in the decomposition of hydrogen peroxide and in the oxidation of ferrous ions by O2 in aqueous solutions.
    The term ‘‘superoxide’’ has been used by many researchers who assumed an exceptional degree of reactivity for O2- especially as a strong oxidant and an initiator of radical reactions.
  • Rajbardhan Mishra added an answer:
    What is the protocol for Reactive oxygen species(ROS) assay especially superoxide when we co-culture, neutrophils with cancer cells.
    Rajbardhan Mishra · Institute Animal Physiology and Genetics AS CR, v.v.i.
    Thank you so much. I hope it will help me.
  • Marcin Nowicki added an answer:
    Does anyone know simple protocols for analysase, superoxide, dismutase and ascorbate peroxidase enzyme activity in plants?
    The papers do not mention standard concentrations to be used for measuring enzyme activity, could someone help me with that?
    Marcin Nowicki · Research Institute of Horticulture in Skierniewice
    Nature Protocls has it all. I highly recommend it (successfully used an Ascorbate microtiterplate assay few years back).

About Superoxides

Highly reactive compounds produced when oxygen is reduced by a single electron. In biological systems, they may be generated during the normal catalytic function of a number of enzymes and during the oxidation of hemoglobin to METHEMOGLOBIN. In living organisms, SUPEROXIDE DISMUTASE protects the cell from the deleterious effects of superoxides.

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