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Lipid Peroxidation - Science topic

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I would like to test for lipid peroxidation (ferroptosis-induced) in leukemic cells using flow-cytometry analyser BD FACSCanto. Now on the analyser, which parameters do I use for analysis of lipid peroxidation?
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Hi Filippus,
If you are using a lipid peroxidation sensor that changes its fluorescence from red to green upon peroxidation, you can use the BD FacsCanto and FITC and PE channels.
Have a look at this kit from Abcam:
KR
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I want to include AA/Vit. C as a positive control for my antioxidant study using Vero cells to compare with my drug compounds. I will measure MDA (lipid peroxidation marker), SOD, and Catalase activity.
My questions:
  1. How do I prepare the stock solution of AA and what are the proper storage conditions?
  2. What is the usual concentration of AA used for in vitro study?
  3. I read AA is light sensitive and unstable in an aqueous solution, so if I add AA in culture media as treatment and incubate 12 to 24 hours, will it work? How to solve this problem?
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1. How do I prepare the stock solution of AA and what are the proper storage conditions?
Since ascorbic acid is highly unstable, you will have to prepare a fresh solution in water each time you perform your experiment unless and until you are using the stable form of ascorbic acid.
You may prepare ascorbic acid in an amber-colored tube or use an aluminium foil to wrap the tube in case you do not have an amber tube since AA is light sensitive.
If you use 2-Phospho-L-ascorbic acid trisodium salt which is a stable ascorbic acid derivative used in cell culture, you may make a stock solution in distilled water and store at 4 degree C for a maximum of 6-7 days.
2. What is the usual concentration of AA used for in vitro study?
The effect of ascorbic acid may greatly vary depending on cell type. Many of the effects of ascorbic acid are observed in primary cell lines. Cancer cell lines and other immortalized cells, however, often show cytotoxic effects in response to ascorbic acid addition that are not observed in primary cell lines. This may be the result of adaptations that have accumulated in these cells due to the culture shock that alters the normal physiological responses to stimuli, possibly involving iron dysregulation or aberrant cell signaling responses.
The concentration of AA to be used for invitro study will depend on the cell type, culture condition (cell culture media) and the experimental design. Usually, AA is used in the concentration range of 5 - 200uM. Sometimes, you may find concentrations up to 100mM being used for invitro studies. Persistent oxidation of ascorbic acid will continuously generate dehydroascorbic acid and breakdown products, such as oxalate and threonate which may have a negative effect on the cells. For example, oxalate has been shown to exert cytotoxic effects, and threonate can impact cell signaling pathways. Please bear this in mind when you decide on the AA concentration for invitro study.
3. I read AA is light sensitive and unstable in an aqueous solution, so if I add AA in culture media as treatment and incubate 12 to 24 hours, will it work? How to solve this problem?
Culturing cells with AA requires control over many aspects of the media and culture conditions. The composition of the cell culture media plays an important role in the rate of ascorbate oxidation. For example, in serum-free RPMI medium the half-life of ascorbate is about 1.5 hr. However, a more rapid loss of ascorbate has been noted in other cell culture media formulations such as MEM including more complex solutions containing serum. Monitoring ascorbate levels and limiting oxidation may not be sufficient to fully recapitulate the physiological roles of ascorbic acid. While redesigning cell culture systems to support biologically relevant reactions of ascorbic acid and eliminate artifacts may limit the practicality of experimental designs.
How to solve this problem?
Ascorbate levels can be maintained in cell culture media by frequent addition of ascorbic acid because the persistent oxidation of ascorbic acid will continuously generate dehydroascorbic acid and breakdown products, such as oxalate and threonate. I suggest if you are going to use 12hr incubation period, carry out a 3hr incubation with ascorbic acid, followed by a wash with PBS and a further 3 hr. incubation with ascorbic acid at the same concentration at 37 degree C in the dark. Continue another cycle of 6 hr. in a similar manner. So, the four consecutive 3hr treatments would be necessary to allow for the possible instability of ascorbic acid in solution.
You could refer to the article attached below for more information on this topic.
Best.
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I made a 20% TBA solution, and after stirring it enough, I can still see it settles at bottom.
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Addition of DMSO (Dimethyl Sulfoxide).
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I used the brain, plasma and serum sample of rats. Preparation of the samples were done on ice bath and centrifugation done at 4 C, then the supernatants were kept in -20 C. the test was done with in 7 days. The brain samples gave satisfactory results on lipid peroxidation test (using 20% TCA and 0.67 TBA). However, plasma and serum samples did not produce any color at all, what cause the absence of colour in these samples?
ps: I also have tried different preparation for the plasma, using citrate and EDTA.
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I have unexpected results in my project where the positive control group had decreased lipid peroxidation levels in comparison to the negative control group. I was thinking that a way to explain was considering that my study was in adult mice and the endogenous antioxidant enzymes are strong enough to hold agains the ROS, also the administration was subcutaneous.
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It was in brain tissue, I explanied the results based on the theoretical assumption that catalase converts ethanol to acetaldehyde and in an acute exposure the enzyme is more expressed and it outweights the convertion by CYP2E1, the higher expression could mean that the enzyme can exert also antioxidant properties, theoretically could be possible. Thanks for the answers
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Dear all,
I'm considering which assay to use for measuring lipid peroxidation in the neuroblastoma cell line. I came across these 3 options: TBARS, MDA assay, and ELISA. TBARS seems less specific compared to MDA assay and ELISA, because TBARS detects anything that reacts with thiobarbituric acid, whereas MDA and ELISA reacts only to Malondialdehyde.
However, I found it strange that from the papers I read, it seems that TBARS is commonest, followed by MDA assay, then lastly ELISA? I was wondering if this observation that TBARS is commonest is true, and if so what's the reason behind? Is it just the price? But I don't find TBARS kit being a lot cheaper than MDA assay and ELISA... Is it then the easy of operation, and the consistency of results?
I would also be grateful for any tips while working with lipid peroxidation / oxidative stress assay, assay recommendations and the like.
Thanks in advance for your suggestions.
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Hydroxyl radical attack on unsaturated fatty acids of phospholipid components of membranes produce malondialdehyde (MDA), a lipid peroxidation product. MDA and TBARS levels provide a significant clue to the magnitude of oxidative stress under disease conditions. Better to opt both rather than ELISA.
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Lipids are susceptible to oxidation when exposed to free radicals. Polyunsaturated fatty acids are particularly vulnerable to lipid peroxidation resulting in cell damage. What are the harmful effects of lipid peroxidation on health?
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This dye itself has color of PE-Cy5 and after interaction with the lipid ROS, it emits green fluorescence. I have also found that this dye also interferes with the PE, PE-Cy7 channels somewhat. So I was wondering if there is an alternative to this dye? (Also, why is this the standard dye for ferroptosis related lipid peroxidation?)
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HNE
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I have searched some research articles related to Ferroptosis. Many kinds of experiments have been done. like, staining, GSH assay, lipid peroxidation assay, etc.
So,
1. What methods (both basic and confirmatory) should be followed?
2. Most of the researchers have been used in the kit. Any method is available other than using the kit?
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In general, evaluation of lipid ROS in cells is the most common way for ferroptosis detection. For this purpose, you can use Liperfluo, C11-BODIPY 581/591, or MDA assay kits. Moreover, you can assay cell viability after using ferroptosis inhibitors, such as ferrostatin-1, liproxstatin, and α-Tocopherol to confirm the occurrence of ferroptosis.
Based on your research you can also assay GSH or iron levels, which is usually optional.
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Hi everyone
i did this protocol to measure the anti-lipid peroxidation activity of plant extracts.
The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. Briefly 100 μL of bovine brain extract sigma (B1502) was mixed with a reaction mixture containing 30 μL of buffer, (100 μL) of (1- 1000 ug/ml) extract or positive control vitamin C , and 30 μL 5 mM sodium nitroprusside (SNP) as the prooxidant. The volume was made up to 300 μL by water before incubation at 37°C for 2 hrs. The colour reaction was developed by adding 300 μL 8.1% SDS (sodium dodecyl sulphate) to the reaction mixture, this was subsequently followed by the addition of 500 μL of acetic acid (pH 3.4) mixture and 500 μL of 0.8% thiobarbituric acid (TBA). This mixture was incubated at 100°C for 1 hr. Thiobarbituric acid reactive species (TBARS) produced were measured at 532 nm.
source:
The problem is that the generated pink colour of chromogen increases with increase the positive control concentrations (vitamin C). where the control the brain extract + sodium nitroprusside (SNP) as the prooxidant gives the lightest pink colour.
Any suggestion?
Thanks in advance...
Maryam
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In addition to the previous answer given by Ayad Palani , you can try to use another system to generate free radicals and cause lipid peroxidation, such as Fe2+ + H2O2. Take a look at:
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We stain our leaf samples of control and stressed with Schiff reagent. However, I did not get good results.
Please suggest other staining methods for lipid peroxidation.
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Dear Javed, thank you very much for asking this interesting question. In this context please note that you can check the answers given to numerous related questions which have been posted earlier on RG. Cited below please have a look at all RG questions and answers related to the topic "lipid peroxidation":
Also please go through the following potentially useful reference describing a protocol for the use of 3,3′-diaminobenzidine (DAB) staining:
Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves
This article has been posted as public full text on RG so that it can be freely downloaded as pdf file. In this context, also please see the answer given to the following question:
How do I DAB stain Arabidopsis Roots?
(5 answers)
I hope this helps. Good luck with your ongoing research and best wishes, Frank Edelmann
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Hi, I am working on HK-2 cells and want to check for MDA accumulation after treatment with our compounds of interest. I am using MDA assay kit from Sigma. However, the kit does not mention about whether the lysate can stored or not. I wanted to know if we can store the cell lystae in MDA lysis buffer of the kit. If yes, at what temperature and for how long? Also, can we use this lysate to measure protein for normalization across samples?
Thank you
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Store the lysates at -80 deg C till you want to use it for MDAa asay.
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I am analyzing clam tissues for lipid peroxidation using the TBARS protocol. To extract lipids for MDA analysis, is it necessary to use PMSF when I am not interested in protein analysis?
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Not sure which article relates to Popovic et al. 2013 (Google has many articles of same author name - Popovic et al. 2013). I can only suggest if you send the article or give the link.
Anyhow, since you are quantifying LPO in clams, I suggest Niehaus & Samuelsson OR Ohkawa method (Both PDF's attached). These two do not use PMSF
PS - Once again about Popovich et al 2013 - Till I read the article, I cannot suggest the utility of PMSF for LPO method.
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Hi
I'm looking for adaptations of Hodges et al., 1999 essay for MDA quantification to microplate, has anyone aware of this? The initial paper is naturally in cuvettes, and most papers don't go into much detail about their methods, becoming dificult to know if this is being done or I'll just have to try it for myself.
Thank you!
Cheers, Luís Pereira
PS: My target are brown seaweed, in case anyone has specific opinion about this.
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Dear Luís Pereira
Good Day .... Hope You are doing well.
The quantification of MDA and all Oxidative Enzymes were fully described in my PhD Thesis PATHOLOGICAL RESPONSES TO INTRATRACHEALLY INSTILLED POLYCYCLIC AROMATIC HYDRO CARBONS AND EFFECT OF CURCUMIN TOWARDS THESE RESPONSES IN SPRAGUE DAWLEY RATS".
You can access it through website of University Putra Malaysia.
Good Luck & Best Wishes.
Keep Safe.
Karim
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Quantifying the level of Malondialdehyde (MDA) is a well established method for detecting lipid peroxidation. Sometimes data is expressed as MDA produced per mg or gram protein. Since we are measuring the level of an aldehyde (MDA), then what is the reason to express the date in per mg or gram protein.  
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I am thik because MDA is an nty oxidant enzyme thus it can elevate then decrease acording to the status of cell ,so thus it in a trace concentration from total amount of proteins
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I am looking for a skilled personnel in Kuwait University that is familiar with antioxidant studies using ELISA kit or other techniques on rat's brain homogenate, particularly the hippocampal and anterior cortex regions.
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Hi Ahmed. I used to work with antioxidant assays on brain homogenates but not with commercial kits. The assays that I used are affordable and pretty much reproducible.
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Hey guys,
Can anyone suggest best 4-HNE assay kit to measure lipid peroxidation in adherent mammalian cells?
Thank you,
Anita
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Hello, I'll renew the topic I'm looking for a fluorescent hne test. Does it exist?
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Dear all,
I followed the specification of Lipid peroxidation (MDA) assay kit from Sigma, and performed MDA assay. But I did not get any pink-color, I got some white sediment instead. Do any of you have the same problem or do you have any idea about that? The MDA standard worked and I can see pink-color, so the procedure of my protocol should be fine.
100% followed the protocol from Sigma. I harvested cells in 10cm dishes.
Best regards
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They may be precipitated proteins.
We usually carry out the test in conical tubes of 600 uL and after incubation, we centrifuge them, we recover the supernatant, and in that solution, we make the readings.
Good look.
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Hello everyone! I want to test if my synthesized compound inhibit reaction of lipid peroxidation. I have searched for protocols in published literature but aren't clear enough since specific time was not mentioned. I found methods where sodium linoleate and AAPH were used. Is that suitable method or can I determine inhibition by TBARS method? Thank you in advance
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Hello
Your question is not clear enough.
If you want to assay antioxidant capacity of your compounds, you may do it by in vitro or in vivo method. For in vitro, you can do DPPH and ABTS. For in vivo method you may use a model animal (mouse, rat, zebrafish,...) and treat it with the compound and an oxidant agent (heavy metals, pesticides, hydrogen peroxide ...). Then measure TBARS levels. You must optimize the antioxidant and pro oxidant concentration and time needed to find their effects.
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Hello everyone,
I would like to measure MDA levels on keratinocytes using a lipid peroxidation assay, I have searched for protocols in published literature but aren't clear enough since I have never performed this assay. Also, if anyone has performed this assay, can you provide me with the catalog number of the kit you have used?
Thanks in advance,
Luveni
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In brief; to 150 µl of sample the following was added: 1 ml trichloroacetic acid (TCA) 17.5%, 1 ml of 0.6% thiobarbituric acid , mixed well by vortex, incubated in boiling water bath for 15 minutes, then allowed it to be cooled ..
Then 1 ml of 70% TCA was added, and let the mixture to stand at room temperature for 20 minutes, centrifuged at 314 x g for 15 minutes, and the absorbance of the supernatant was measured at 532 nm.
Guidet, B., Shah, S. V.(1989). Am. J. Physiol. 257(26):F440.
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Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), have been utilized to reduce lipid peroxidation in food products. However, I couldn't find a report on these chemicals as active compounds to reduce oxidative damages in human body when the living organisms unable to develop their own antioxidant defense mechanis.
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Thank you!
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I want to determine lipid peroxidation rate from insect larvae.
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I am conduction some bio-assays on the digestive tracts of bivalves exposed to a series of concentration of the same chemical, along with keeping control samples (cannot mention chemical or detailed results due to data concerns). In short, after testing and statistical validation was completed we have an odd result:
Glutathione S-transferase (GST) assays show samples had no statistical difference in activity between control samples and any samples exposed to the chemical, and only a mild rise in average activity that can't be thoroughly distinguished between control and chemical exposed samples. Meanwhile other tests (Lipid peroxidation, DNA damage, FRAP, etc) all show a clear difference in response, with the chemically exposed samples all showing rises in stress responses. This result seems quite odd, as GST is often the starting indicator of whether there is any chemically induced oxidative stress, and the next few assays often help to pin down where and why the stress is being caused. I know that this assumption (GST = all stresses) is not guaranteed but it is fairly common practice to use it as such.
My only real idea is that in general the mussels remained stressed even after 14 days of lab storage to purge at optimal temperatures, media cleaning and regular feeding. And as such the GST remained raised on all samples from general stress from their new environment, but then the other assays showed how the added chemical distinctly rose the stresses in more specific ways. Does anyone else know of other possibilities as to why GST would remain indistinct between control and chemical exposed samples, while other assays would show a clear rise in stresses in chemically exposed samples?
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I agree with Dr. María Antonieta López Hernández
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I tried to run a lipid peroxidation assay with DPPP , the plate in black with clear bottom. I read the fluorescence from top and since my samples are diluted in PBS I used put it in one well and use the read as blank. But everytime I read the plate I have different results. Is there any special consideration for running this type of essays in terms of wells localization? time?
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The 0.01% Triton X-100 concentration is below the critical micellar concentration, so it should not sequester the lipids in micelles. It is sufficient to coat the surface of the plate, leaving a hydrophilic layer. You could also try using so-called non-binding surface (NBS) plates, but they are more expensive than untreated plates.
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Dear colleagues;
What are the novel and accurate methods for testing the following:
  1. Total cholesterol conc.
  2. Triglycerides conc.
  3. HDL-C conc.
  4. LDL-C & VLDL-C
  5. Lipid peroxidation marker (MDA)
Best regards;
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Hello, see the info below you can find some news there.
Good luck!
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I would like to analyze lipid peroxidation (TBARS COLORIMETRIC METHOD) in plasma samples of rats. But I don´t getting satisfactory results with the current technique that I possess. So, I'm looking for an alternative technique for the colorimetric spectrophotometric performance of this analysis in a 96-well microplate.
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Try preparing the TCA-TBA-HCl stock consisting of 15g trichloroacetic acid, 0.375g thiobarbituric acid in 100 mls of 0.25N HCl
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Hello, I am trying to induce oxidative damage in neurons (derived from IPSC) by using hydrogen peroxide.After treatment I want to see if there is any evidence of lipid per oxidation (by looking for malondialdedye) or if there is any evidence of 8oxoguanine accumulation. can anyone advised how long I should wait after treatment before assessing for these?
thank you
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I wonder how you would determine accumulation of MDA ex vivo while you may not have info regarding baseline/level of total antioxidant (TA) present, it is virtually indeterminable!
Be reminded that;
1. MDA development is a function of how much TA already present. This could only be determined by taking baseline, I wonder how you could this in animal models with limited blood supply.
2. MDA begins to increase immediately you induce with radical-generating substances, only curve may inform you proper how fast this takes place, consequent upon TA concentration in the body which is a function how sufficient nutrients had been earlier uptaken to the system.
Finally, I advise;
1. Search for literature to see previous procedure in doing this.
2. Or set a timeline after overloading with peroxide, and report it so-so hourly e.g injected 4-hourly for 4 dates, depending on your topic. Be informed that it takes much time before reactive oxygen species from lipid peroxidation do damage to DNA and initiate, promote or progress cancer.... et cetera.
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Hi
Can anyone give me correct protocol for TBARS assay to measure lipid peroxidation in Supernatant of MCF-7 cell line?
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thank you
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Dear Researchers
I am studying the mechanism of lipid peroxidation in tomato fruit. It would be appreciated if anybody help me sharing the detailed procedures of measuring MDA, hydrogen peroxide and superoxide content in fresh tomato tissue using spectrophotometer.
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Dear Dr. Shafiq,
These 2 papers are for MDA protocol.
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I want to do a fluorescence measurement (96 well) after a lipid peroxidation assay on Mia PaCa-2 cells using c11-BODIPY. Could anyone share a protocol with me.
Do I have to pretreat the cells with BOBIPY before my peroxide inducing agent or vice versa.
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Thanks Nasrin
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I am currently doing analysis of antioxidant enzyme activities in rat liver tissue. I would be glad to get current reference on antioxidant enzymes and lipid peroxidation assays
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Dear Abdelrahim, in my more than 50 years experimental work, I came to conclusion that you better avoid any kits. They are for lazy or inexperienced people. With a kit you have no control what you do. Believe me. It is much more reliable and much, much cheaper to establish a method by yourself.
Sincerely,
Alexander.
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I am currently attempting to run a TBARS assay on algal samples under different stress conditions. I have been following the Hodges et. al, 1999 protocol from their paper, Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. I am using a spectrometer to measure the MDA equivalents but I am having difficulties getting the end quantitative values for all absorbance readings. When i perform a technical replicate on the same cuvette my absorbance values changes quite drastically. They usually increase but some do decrease. Is this reaction light sensitive? Am i detecting other compounds which increase in response to the spectrometer? I don't know how to get around this. Thanks in advance
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Hi, what differences in absorbance do you observe?
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Hi!
We are trying to estimate lipid peroxidation in leaf tissues of the seagrass Phyllospadix torreyi, following the protocol from Hodges et al. 1999. But this protocol does not work (i.e. we do not obtain the characteristic pinkish-red chromagen). This is so weird, because we have used this protocol with other seagrass species and land plants, and it always worked in the past!. Furthermore, at the same time, we are using the same protocol and reactives for seaweeds in our lab, and we are obtaining fantastic results. Could anybody kindly provide me any explanation or could suggest some modification for this protocol? Thank you very much!
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thanks a lot Marco. I think you have already done enough! Yes...this seagrass is my particular nightmare! :)
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I want to determine MDA content in tomato leaves under salt stress conditions. For this purpose I have collected leaf samples after completion of different levels of stress treatments as well as foliar spray of amino acids. I preserved the leaf samples in 10% TCA and kept in freezer at -40 since 2 weeks. Now, when i started my experiment by following Health & Packer protocol, I am not getting any color at the end of reaction. All reaction mixture are transparent not showing any color (must be some shade of pink) when we put at water bath for 25 mints. Firstly I was thinking that may be some issue in samples than I got some fresh leaf samples and perform the same procedure but no result . All the chemicals used here are new so I am not getting the point? Could anyone please suggest me that what can be issue and guide me. Thanx
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Hi Zaib un-Nisa,
Have you able to solve your problem? I am trying to measure lipid peroxidation in leaves of a seagrass (Phyllospadix)by using the protocol from Hodges et al. 1999, and I have the same issue (I cannot see the pink reaction).
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I'm reading about the TBARS assay and I want to know for what TCA is used in this assay? What is the purpose of adding TCA in the sample?
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TCA precipitates macromolecules.
Assay of TBARS (thiobarbituric acid reactive substances) measures concentration of malondialdehyde produced due to degradation of unstable lipid peroxides.
TCA has two functions first dehydrating the hydration shells around the protein by which the anionic TCA triggers partial protein unfolding through disruption of the electrostatic interactions by determining the native tertiary structure of the protein. TCA is less effective for disordered proteins.
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Dear Scientists,
I am looking for a method to isolate and analyze reactive oxygen species (ROS)-damaged proteins. I know that there are kits to detect individual ROS such lipid peroxidation, protein carbonylation and others. But, I am searching for a method to combine all of these in one! Have you heard of such a method?
Thank you 
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Dear Assraa Hassan Jassim,
I assume that you are informed about the duration of the procedure of determination of protein carbonylation. Concerning the lipid peroxidation, I would like to cite a known opinion about it:
"In contrast to lipid peroxidation the products of which typically appear after a lag time, protein damage by reactive oxygen species takes place directly and immediately. The most common drawback of protein degradation assays is the complexity of the applied methods such as radioactive or fluorescent labeling, gel electrophoresis, Western blots, or immunoprecipitation".
(Pacifici, R.E., Davies, K.J.A.: Protein Degradation as an Index of Oxidative Stress. In: METHODS IN ENZYMOLOGY, 1990, Vol. 186, Part B, Eds. Packer, L. and Glazer, A.N., Academic Press, New York, p.p. 485-502). We developed a method, based on two different measuring principles, which is free of these disadvantages (US patent 7,125,723 B2 and two publications are attached). The preparation of a blood sample for determination of the oxidativen modification of plasma proteins lasts 10 minutes, the measurement 2...3 minutes. The detection of oxidative modification of nucleotides and nucleic acids is possible too (4th attachment).
With best regards,
I. Popov
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Any suggestion what I should change?
I'm trying of 2kV/cm ; 4 kV/cm; 10 kV/cm PEF treatment. What concentration of cells I should use?
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Firstly, make sure that the device is well calibrated then you can try the highest concentration.
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During the analysis of lipid peroxidization (MDA assay) as a stress bio marker under abiotic condition in microbial cells by manual method -
1. is it necessary to carry out the Standard curve ?
2.If yes, MDA (commercially available) should be used as a standard or any other alternative standard is available?
3. Which solvent is to be used for dissolving TBA - a. TCA or b. NaOH ?
4. how to calculate (formula) the concentration of MDA in the sample after assay ?
Thank you
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3. 1 % TBA (w/v): 1 g TBA dissolved in 100 mL of 0.005 N NaOH
4. MDA values in nmole were determined with the extinction coefficient of MDA - TBA complex at 532 nm (e532= 1.56 x 105 L/mol.cm). TBA reactivity was expressed per mg of protein.
Jain and Levine (1995)
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I am using Reilly and Aust, 2001 method
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Comparative Biochemistry and Physiology, Part C 159 (2014) 10–21:
Lipid peroxidation in hepatocytes was estimated using the
thiobarbituric acid reactive substance (TBARS) assay as previously
described (Hermes-Lima et al., 1995) with slight modifications. Briefly,
cells were resuspended in twenty volumes of ice-cold 1.1% phosphoric
acid. Then 0.2 mL of homogenatewas mixed with a medium containing
1% thiobarbituric acid, 50 mM NaOH, 0.1 mM butylated hydroxytoluene
solution and 0.1 mL 7% phosphoric acid. After boiling for 15 min
the samples were cooled on ice for 10 min. Ice-cold butanol (0.5 mL)
was then added and the samples were thoroughly mixed and centrifuged
for 5 min at 10,000 g. The top butanol layer was then used for
spectrophotometric measurements (532 nm). Malondialdehyde standards
were prepared in 1.1% phosphoric acid and treated as samples.
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I'm hoping to use the same (currently frozen brain) sample to measure GSH/GSSG/Total as well as MDA assay - what would be the best way to prep this sample?
I'm using the sigma kit for MDA and they suggest homogenizing in the lysis buffer containing BHT + Perchloric acid (150uL, 2N/10mg tissue).
The GSH/GSSG/Total kit suggests using 5uL of 6N perchloric acid/10mg tissue.
Could I potentially prep the sample as for MDA assay and use the same supernatant to measure glutathione too?
TIA!
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Please have a look at enclosed documents...hope you like them...
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I want to determine lipid peroxidation markers only in area 1 hippocampus in rat,. Can i?
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Hi Ghalia,
You can take a tissue punch, either from the whole brain on a cryostat after slicing down to the CA1 region, or from a slice or set of slices. Doing it from a cryostat is probably easier and faster. Use a brain atlas to figure out where you want to punch, how deep you want to go, and the diameter of the sample you want. You should be able to get plenty of tissue for whatever assay you use.
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I have read that for lipid peroxidation we can use DPPP, alexa fluor 488 or Bodipy 581/591 C11, but I can't find specifically for Saccharomyces cerevisiae.. Can I use the same dyes for them? Maybe there are some articles about that? 
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I think you may use fluorescent dye C-11 BODIPY 581/591.
Best of Luck!
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with detail of procedure
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In fact, MDA measurement is one of the easiest way to assess lipid peroxidation.
Many technics were develepped. The better are HPLC with flurescence detection.
If you can't, you have to choose fluorometric measurement. Avoid absolutely colorimetric technics. They suffer of many interferences. If you have possibility use synchronous fluorescence. So, you can strongly increase specificity. I published the following paper : " Improved fluorimetric determination of malonaldehyde.", Clinical Chemistry, 1991, 37/7, 1273-75 .
best regards
marc
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hi, i need recent references for the estimation of Collagen which is similar to the protocol described by Woessner (1961), DNA similar protocol described by Burton, (1956), Lipid peroxidation similar to Buege and Aust (1978) and western blotting.
Thank You
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thank you Dr.amin and Dr michel
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I am trying to make standard for MDA for lipid peroxidation plz suggest me a suitable protocol or reference paper for it.
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Dear Colleague,
The determination of malondialdehyde (MDA), widely used as biomarker of lipid peroxidation was usually performed according to the method of DRAPER. & HADLEY (1990):
 DRAPER, H.H. & HADLEY, M. (1990).- Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol., 186, 421-431.
 See attached some publications dealing with MDA. It can help.
 Best regards Pr. N. Soltani
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To perform Lipid peroxidation inhibition assays in egg homogenates, I have faced difficulties to make proper egg homogenate. It was difficult for me in pipetting the sample. Please somebody guide me the proper way to  prepare 10% (v/v) egg homogenate.
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thank you sir..
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Hi every one I need more information about how low temperature can affect oxidative stress parameters in fresh water fish
 bet regards
Safia Habila
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Interesting question Safia..
Extreme Temperature Responses, Oxidative Stress and Antioxidant Defense in Plants (DOI: 10.5772/54833)
The cellular changes induced by either high temperature  or low temperature  include responses those lead to the excess accumulation of toxic compounds, especially reactive oxygen species (ROS). The end result of ROS accumulation is oxidative stress]. In response to high temperature  the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) can lead to the production of H2O2 as a consequence of increases in its oxygenase reactions . On the other hand, LT conditions can create an imbalance between light absorption and light use by inhibiting the activity of the Calvin–Benson cycle. Enhanced photosynthetic electron flux to O2 and over-reduction of the respiratory electron transport chain (ETC) can also result in ROS accumulation during chilling which causes oxidative stress. Plants have evolved a variety of responses to extreme temperatures those minimize damages and ensure the maintenance of cellular homeostasis. A considerable amount of works have explored that there is a direct link between ROS scavenging and plant stress tolerance under temperature extremes . Thus, the improvement of temperature stress tolerance is often related to enhanced activities of enzymes involved in antioxidant systems of plants. Plants exposed to extreme temperatures use several non-enzymatic and enzymatic antioxidants to cope with the harmful effects of oxidative stress; higher activities of antioxidant defense enzymes are correlated with higher stress tolerance. Different plant studies have revealed that enhancing antioxidant defense confers stress tolerance to either  high temperature  or low temperature  stress .
Like annual crops, perennial crops are also sensitive to extreme temperature. Fruits and nut trees are important crop plants which often face extreme temperature stress induced damages. Every fruit tree species has a range of optimum temperatures  above or below which the growth and yield markedly reduced. The mean temperatures range for optimum growth of most tropical fruits are about 24-30°C . For instance, mango (Mengifera indica) tree can tolerate high temperature up to 48°C only for a certain period of time , on the contrary it has only partial tolerance to low temperature  In another study, Schaffer et al. observed that monoembryonic mango cultivars tend to be more low temperature  tolerant than polyembryonic cultivars . However, several studies have shown that low temperature promote reproductive morphogenesis in mango. Dinesh and Reddy studied the responses of fruit trees to temperature and observed differential responses to temperature in different fruit species. They concluded that lychee and longan require a warm sub-tropical to tropical climate that is cool but also frost-free or with only very slight winter frosts not below -4°C, and with high summer heat, rainfall, and humidity. In longan, stressful temperatures of <15°C at the young fruit stage reduce fruit growth potential and final size as reported by Young et al.. Stressful  low temperature  also induces excessive fruit drop and severe fruit cracking ....
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I’m planning to investigate the toxicity of MDA for cell line. Has anyone know how I can synthesis Malondialdehyde & analysis the MDA standards by TBARS. Also, is there any book has related information's with MDA.
My reagent information is:
TMP: MW=164.20 g/mol, d= 0.997g/mol, 99%.
TBA: MW= 144.15 , 98%
Kind Regards,
Hiba
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Hello Hiba,
The concentration of MDA in tissue or cell homogenates can be obtained by the TBA-RS technique by performing a calibration curve of known concentrations of MDA. MDA can be bought, why do you want to synthesize it?
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Salt stress experiment by in vitro culture.
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8-iso-PGF2α (8-isoprostane) a stable prostaglandin-like product
formed from arachidonic acid by the direct action of ROS
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whole antioxidants are required. However protocol which include the majority or main antioxidants are preferred.
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Thanks to all. It was great help
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In such diseased conditions I tried to examine the effect of plant extract and I would like to assess the changes in ACh concentration.
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Dear Dr.Jabbar
     I think good link by Dr.Hiba
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How can I prepare the standard curve of Malondialdehyde (MDA) especially with Tetraethoxypropane (TEP) for TBARS calculation in UV spectrophotometer. Can anyone suggest how should I prepare the standard solution of TEP and in what range ?
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Dear Dr Carpenter,
May lab protocol was as follows in my recent submitted work:
2.9 Estimation of lipid peroxidation (malondialdehyde (MDA) assay)
The magnitude of lipid peroxidation was determined by measuring MDA which is thiobarbituric acid reactive substance (TBARS). To precipitate the proteins, TCA (30%; 0.5 ml) was added to tissue extract (0.5 ml), vortexed for 30 seconds, and finally centrifuged at 3,000 rpm for 5 minutes. Thiobarbitoric acid (TBA; 1%; 500 µl) solution and 500 µl of distilled water were added to the supernatant and the resulting mixture heated for 1 hour at 98 °C, then cooled to room temperature and kept in ice for 5 minutes. At last, the absorbance of pink mixture recorded at 532 nm. Standard graph was plotted using 1,1,3,3-tetraethoxy propane (TEP) to estimate MDA values [30].
[30] Ohkawa, H., Ohishi, N., Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 1979,95:351-8.
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Now, i try to assess the MDA and antioxidant levels like SOD, gluthaione and catalse in human sperm. I used 0.01% Triton-x and centrifuge for 6000 g, then collected the supernantant to assess the MDA levels. But the MDA level it very low (less than 2 nmole). Has anyone recommend the best protocol to isolate supernatant in human sperm? Thank you for your kind suggestion. 
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Just after ejaculate collection, let it until liquefaction. Close to 15 min. Then centrifuge at low rotation speed, below 2000 rpm, at +4°C. Separate, rapidly, semen from spermatozoa pellet. And you can freeze it directly.
To measure SOD and catalase enzymatic activities, MDA concentration, avoid triton. Use crude semen.
But to measure GSH and GSSG you have to add to semen perchlororic acid (PCA) to stop glutathione degradation. The protocol I use is the following. to 100 microliter of diluted (1/2) seminal fluid add 50 microliter of 2% PCA. Vortex. centrifuge high speed (10000 rpm). then freeze supernatant until experiment.
MDA level is low in semen.
sincerely
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I have synthesized magnetite nanoparticle and coated them with protein to render biocompatibility, I have already performed MTT assay and now planning to go ahead with ROS detection assay? How should I perform the assay, by detecting lipid peroxidation or is there any other source?
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If you are trying to estimate the lipid peroxidation induction capacity of you protein coated magnetic nano particle, then the attached article can help you to design your experimental protocol. Hope this will work for you.
Thank you
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I want to measure lipid peroxdation level in mouse colon tissue homogenate. Can anyone help me out in this? 
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I think this article will help you to sort out your problem. To estimate lipid peroxidation in tissue homogenates, use tissue homogenate insteed of using blood. Rest you need to follow the same protocol. Feel free to ask questions regarding lipid peroxidation.
thank you
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Hello all,
I have plant treated with different concentrations of copper. Interestingly, the callus exhibited increased levels of MDA, despite enhancement in their biomass. There was stimulation in some antioxidant enzymes such as SOD, POD and GST. How can you explain this? Have you faced this before?
Most paper that discuss heavy metal mediated-oxidative stress, particularly copper as a transition metal, relate the inhibition in growth or biomass to the increase in lipid peroxidation concomitant with inhibition or even stimulation in the antioxidant defense system, in contrast to my results. 
I know that among the strategies the plant may employ is to repair the degraded membranes and refold the unfolded or misfolded proteins, but I am not sure if that is the situation for my samples.  If it is, should it appear as decrease or increase in lipid peroxidation?
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Thank you Afyaa, hope you good luck in your research.
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I did antioxidant analysis which was catalase. My samples were rats liver. How can i proceed to analyse the end results if the absorbance rate was more than 1.2 (as suggested in the kit manual)? The manual suggested to dilute the samples, however all the reagents were almost finished. Need advice on this. Many thanks for your help. 
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What kind of detector you used?  A simple way is to use other wavelengths, which have lower absorptivity coefficients. 
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cell line sample to working the antioxidant property so reduces on error.
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You can monitor oxidative stress generated in the cells using CellROX and MitoSOX stain. CellROX can be used to monitor nuclear and cytosolic oxidative stress (or ROS) while MitoSOX (superoxide indicator) is specific for mitochondrial oxidative stress. You need to perform fluorescent imaging experiments after the treatment. We used SH-SY5Y, MCF-7, BV-2 and RAW cells.
Protocol for CellROX stain is given below;
Procedure:The human neuroblastoma cell line SH-SY5Y, was grown in DMEM-F-12 (1:1) medium, supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and maintained at 37°C, 5% CO2 in a humidified incubator. Once cell reach 80 % confluence further experiment was performed. Solutions of compounds were prepared in DMEM-F12 media (without phenol red). These solutions were added to the cells and cells were then incubated for 18 hours. After incubation media was removed, CellROX green was added to the wells and incubated for 10 minutes. Then cells were washed with PBS. Cells were fixed using 4 % paraformaldehyde for 10 minutes. Then cells were washed Coverslips were removed from the plates and inverted onto the grease free slides. Slides were sealed with nail polish. Confocal live cell imaging for CellROX was performed using green filters (ex/em= 488/500-550 nm). 
In addition to this. you can also measure NO production in cells (like BV-2 and RAW) spectrophotometrically using Griess reagent. You can find this assays in supporting information of this article (http://onlinelibrary.wiley.com/doi/10.1002/slct.201600808/abstract
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In paper or techniques manual, 7.8 value is taken for calculation of TBARS (mg malonaldehdyde/kg sample). But no one is mentioned how this 7.8 value is considered for calculation. Can anyone enlighten me it properly? 
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Do you find any solution for this question? 
I'm stuck with the same question as well.
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should any blank be used in TBARS estimation from a heat stressed plant sample 
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Dear Om Prakash
1) Firstly, you have to set as zero absorbance your reference blank (for example, in the below protocol the reference blank is 1-butanol) before you execute any sample measurements.
2) Also, you have to subtract from your sample measurements the appropriate sample and reagent blanks. For details see the below Lipid Peroxidation (LP) protocol for plant samples:
The samples were ground at 4oC under dim light (to prevent artificial LP) in a porcelain mortar with homogenization buffer containing 50 mM Na2HPO4 pH 7.2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM butylated hydroxyanisole (BHA) and 0.15% ethanol. The homogenization was carried out using 5 mL buffer per g fresh sample weight. The homogenate was assayed for LP products by a modified thiobarbituric acid (TBA)-based method (Buege and Aust, 1978). Specifically, 0.5 mL homogenate (or appropriate dilution) was mixed with 0.5 mL TBA reagent containing 0.5% (w/v) TBA, 20% (w/v) trichloroacetic acid (TCA) and 0.33 N HCl. To the resulting mixture was added 5 μL 2% (w/v) of the lipid antioxidant BHA (in absolute ethanol) to prevent artificial LP during the assay. The mixture was incubated at 100°C for 15 min and brought to room temperature (RT). To that was added 1 mL 1-butanol, mixed by vigorous vortexing, centrifuged at 15,000 g for 3 min, and the absorbance of the upper butanol layer (determined with a spectrophotometer) was measured against a sample blank (0.5 mL sample mixed with 0.5 mL 20% TCA in 0.33 N HCl and with 5 μL 2% w/v BHA) and a reagent blank (0.5 mL homogenization buffer mixed with 0.5 mL TBA reagent and 5 μL 2% w/v BHA).
As you can see
1) The sample blank must be prepared and measured under exactly the same conditions (quantity of extract, incubation, heating etc.) with the corresponding sample, with only one difference: the reaction solution added to the extract sample must not contain thiobarbituric acid (TBA). Thus, your sample blank will measure only the non-specific absorbance including the natural colour of the extract.
2) The reagent blank (which must not contain any sample) will measure the possible homogenization buffer interference.
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I have to measure the oxidative stress enzymes activities like SOD, Catalase, GSHpx and lipid peroxidation of workers having exposure to chromium from erythrocyte lysates, please recommend me some good protocols how to prepare human erythrocyte lysates?
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Thanks José Ramón Vielma for your help,  the papers you sent really helps me understand the problem better and Thanks further to Surendra Katyare for the paper you sent.
Thanks Again.
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We have measured the activities through enzymatic or non enzymatic assays  of Super Oxide Dismutase (SOD), Catalase, Glutathione and lipid peroxidation from hemolysate, now if we need to calculate the same in serum, do we need to follow the same protocols or there are different protocols for serum. Please help to understand this.
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It is possible to use the same chemical reaction to measure GPx and Catalase activities. But, take care, the seric activities are dramatically lower. Moreover, in function of the test used, you may have some difficulties to measure SOD in sera. Particularly if you use a tetrazolium blue. INT, used in the ransod kit, works  with hemolysate, but doesn't with sera. While WST-1 used in AbCam SOD test works in the two case.
Good luck
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Could somebody provide a good method for analysis of lipid peroxidation in drosophila?
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We use the Tbars methodology and MDA levels by HPLC.
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I know that there exists a direct relationship between lipid peroxidation and SOD, Cat, GPX etc., But there is lot of ambiguity when it comes to working with a particular cell line. Please post your inputs here. Thank you.
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I send my papers about this argument.
Regards
Sebastiano
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I am developing a method to measure antimicrobial death caused by ROS in a bacterial culture.
In particular, I want to measure oxidative stress compared to my reference sample.
I thought of different fluorescent reagents, as would the H2DCF, HPF or Click-iT lipid peroxidation kit- imaging Alexa Fluor 488
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I think this article may be helpful:
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I need to prepare 42mM H2SO4 to be used in MDA assay but I have trouble in finding ways to prepare it. 
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98% H2SO4 → 42mM H2SO4.
Add 2.27 mL of 98% concentrated sulfuric acid to about 700 mL of distilled iced water in a 1 litre measuring cylinder and make up to 1 L.
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I do TBARS assay by following the protocol of Ohkawa et al, 1979 (Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction). I need a clarification on the working protocol this method.
The following is the protocol as per original Ohkawa Paper, i.e.
1. 0.2ml of sample is added with 0.2ml of 8.1% SDS, 1.5ml 20% acetic acid at pH3.5, 1.5ml of 0.8% of TBA
2. This mixture is made up to 4ml and boiled for 1 hour at 95-degree celsius.
3. After cooling, 1 of distilled water and 5 ml of n-butanol & pyridine solution have to be added and shaken vigorously.
4. After centrifugation (4000rpm) for 10 minutes and a supernatant was read at 532nm.
I used to do this protocol by eliminating the adding of distilled water and n-butanol & pyridine solution (The step 3). However, I can get the standard curve with R2 value 0.99. I also performed with 3rd step, but I didn't get the proper standard curve. Please let me clarify that, Is following the original protocol mandatory (with 3rd step) or following this protocol without 3rd step is sufficient.
Thanks in advance for your valuable suggestions.
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All photometric TBARS assays are non specific for MDA because they measure other aldehydes as well, which absorb at near same wavelength as MDA does. To measure specifically MDA you will have to go with fluorometric assays. Such is the following:
Grintzalis, K., Zisimopoulos, D., Grune, T., Weber, D., Georgiou, C. D. (2013). Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides. Free Radical Biology Medicine 59: 27-35.
If interested, you could get a free copy from my Researchgate site
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It is found that vitamin C supplement helps in reducing MDA level in liver disease patient. Further studies suggest that zinc level is reduced in liver patient. So what will be the relation between MDA and Zinc?
Again we know that NO is a lipid peroxidation products. If NO (Nitric oxide) is also increase in liver patient then what will be the relation between zinc and NO?
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Best regards,
Kívia
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I have taken data of both assays.If I want to compare antioxidant activity with relation of Lipid peroxidation(By MDA) then which type of calculation/formula/software will used through which I recognized either antioxidant are working well in this biological system or not.
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Dear Samrina,
I recently worked on antioxidant and lipid peroxidation. You can input your data on excel or you can use SPSS software package program to correlate the your findings about antioxidant and lipid peroxidation profile.
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What happens if lipid peroxidation level decrease in control animal?
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Increased lipid peroxidation is a mark of elevated malondialdehyde content. In control animals it is supposed to be lower than the treated groups provided the chemical used for treatment is a ROS generator.
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I am doing TBARS assay to measure lipid peroxidation in LPS induced RAW 264.7 cells.I am not getting any result as there is no appearance of  pink color in sample after doing assay.I am following this protocol.please find the attached document.
thanks in advance.
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Dear Sinha,
I have followed the attached protocol. It works for me.
Hope for the best.
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I am trying to standardize Lipid per-oxidase assay using TBARS method. At the end of the assay, when I am recording OD of the final pink coloured  product, the reading is fluctuating and is undergoing a gradual reduction with time. 
My questions are:
What should I do to have a stable OD?
Is it OK to consider the first OD as correct one, shown just after placing the cuvette into the Spectrophotometer?
Please help...
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Dear all.
It is due to fact that your pooled organic solvent (butanol), wherein your all TBARS are remaining at the end of reaction, absorb humidity from the environment. So try to heat all of them for another 5-10 min after pooling the TBARS-containing layer, and take OD in a humidity-free room as quickly as possible.
This is the solution.
Good luck!!
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I have seddlings of sorghum and I could not homogenate them at the detected time of incubation, so I conserved them in the refergerator for 2 days, can I homogenate them and estimate lipid peroxidation and H202?
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Procedure and calculation part
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hi,you can be used this articles in your pruject:
1.             De Vos CHR, S. H. De Waal, M. Vooijs, R. E. W؛ 1991. Increased resistance to copper-induced damage of the root plasma membrane in copper tolerant Silene cucubalus. Physiologia Plantarum, 82: 523–528.
2.Qiujie D, Bin Y, Shaobai H (1997) Response of oxidative stress defense systems in rice
(Oryza sativa) leaves with supplemental UV B radiation. Physiol Plant 101:301–308
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I want to carry my plant tissue abroad and it is not possible to maintain the samples in -80 centigrade so I need to freeze dry and carry samples. Will it affect the antioxidant enzyme activity?
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Hi. The best method for preventing of enzyme denaturing is freezing in liquid nitrogen. by freezing we can protect enzyme.
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I am getting different concentration values for the same sample. For liver tissue, at first I got 35 µM concentration. But, after one day the concentration value has come 13.5 µM.To assay MDA from tissue I have followed the protocol below-
  1. Homogenized in Tris-HCl (50 mM, pH-7.4).
  2. Centrifuged at 12,000g for 10 mins.
  3. Taken supernatant.
  4. 50 µl homogenate were mixed with 500 µl of 0.67% TBA, 500 µl 20% trichloroacetic acid.
  5. The mixtures were incubated in a boiling water bath for 20 min.
  6. After cooling to room temperature, the reaction mixture was centrifuged at 4000g for 10 min and the absorbance of the supernatant was measured at 532 nm.
For standard curved I have used 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM MDA dissolved in homogenizing buffer.
Every time I used to make standard curve prior to experiment. How should I troubleshoot my problem? Thanks in advance.
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Dear Debaprasad Koner, 
I would recall the same issues suggested by Pierre, the only difference that in our lab a 20% TCA and therefore TCA/TBA ratios varied from the mentioned  (~1/1) without problem. The issue with dissolving TBA deserves special attention, especially because it also changes the fluorescence levels depending on time and preparation procedure.  In addition to that: 
1- You could consider to add an antioxidant as BHT in your homogenates to prevent further MDA production during sample preparation.