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Hi Hello researchers! need help. I had prepared a cereal-based fermented drink, keeping sensorial properties in mind I centrifuged the sample and collected the supernatant.
And the further analysis was carried out in the supernatant. Antioxidants assays {DPPH/FRAP}. How do I represent the results? Some say it has to be in w/v some say only volume rep eg 500microL but usually in articles we see w/v . And IC50 also is in w/v
So how do I represent this? Thanks in advance....
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the results are converted as mg/100 mL (expressed as dry weight soluble solids fraction by 100 ml in the vol of sample used.
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Hi! I am doing antioxidant assays DPPH, FRAP, and TBARS. I want to know and compare how my samples will react with the mentioned assays. as for the concentration range, should I use the same set of concentrations for all assays? Or can I set different conc range for each assay?
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Usually when you want to compare the effect of two different techniques, you must standardize by a parameter that is constant in the samples, for example the protein concentration in the tissue.
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Hello everyone
I would like to know please what are the in vitro biological activities that I can test for hydroalcoholic plant extracts. Namely that I have already carried out the following analyses: - Antioxidant activity (DPPH, FRAP)
Antibacterial and antifungal activity
Anti-inflammatory activity (test of egg albumin denaturation and BSA denaturation test) Antihyperglycemic activity
Thanks in advance
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Biological activities of plant extracts that some can tested are many. In addition to the one cited above, you also have : anti-tuberculosis a antitumor, anticancer, antimalarial, , anti-inflammatory, anti-aging, anti-proliferative, hypoglycemic, hypocholesterolemic, antihypertensive, induction of resistance activities...................
The above question should not be asked like that !!!!!!
The orientation of the objectives of your project should guide you, on which biologocal activity you can evaluate
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I will be using the solution for the FRAP assay. How is this prepared and how long can it be stored? Is it sensitive to light?
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Hello Duri Eun
The molecular weight of FeCl3.6H2O is 270.2.
So, add 0.0541g of Iron (III) chloride hexahydrate to 10ml ultrapure water.
You may weigh 0.0541g of Iron (III) chloride hexahydrate and transfer the Iron (III) chloride hexahydrate to a beaker of appropriate size. Add 5 mL of ultrapure water to the beaker to dissolve the salt. Transfer your solution to a 10 mL volumetric flask. Using ultrapure water, complete the solution's volume to reach 10 mL. You may prepare this solution on the day of the experiment.
It is better to prepare a fresh solution. This solution is sensitive to light.
Best.
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Can we use FeCl3 instead of FeCl3. 6H2O in FRAP assay?
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Yes. You can use anhydrous ferric chloride. The key point is the equal molar amount of iron.
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For the FRAP assay, I am confused whether I should just get the EC50 or the trolox equivalent values (which is most often used in studies I came across). Can you explain what is the difference and for which analysis fits each one??
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IC50 is used for DPPH method, as Weihao Meng mentioned , the reason is the absorbency because the relation in DPPH between conc. and abs. is not linear its (S shape ) line which don't allow to calculate the conc. .
In FRAP we preface the use calculation the concentration using standard as the relation is linear.
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I made some experiments on plant extracts to look for antioxidant activity using the DPPH and FRAP method. Now I wish to know how to calculate the radical scavenging activity(%RSA) using the FRAP method. Are we using the same formula with the DPPH?
NB: I made standard curves using ascorbic acid for both methods.
Thanks in advance.
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I'm wondering why do you use some methods without reading the descriptions of these methods. You must do a minimal home work yourself before asking for the help.
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Greetings everyone,
I'm currently working on my bachelor's thesis about thermic effects in FRAP(fluorescence recovery after photobleaching)-experiments when using a confocal laser scanning microscope (CLSM).
Right now I'm trying to figure out what causes these increases in intensity around the bleach-spot that starts to shows up when doing FRAP at a certain depth. For all my measurements I set the height as 0 μm where the excitation with the laser caused the highest intensity. The increases in intensity started showing up at a height of 20 μm and persisted up until 300 μm (I went this deep into the sample out of interest). My samples were solutions of rhodamine B in glycerol and sulforhodamine B in glycerol (both with a concentration of roughly 1 g/L).
My research so far didnt give me any possible explanation for this phenomenon. I'd be glad if someone in here could either give an explanation or provide me with literature on this subject. I will add 2 pictures so you know what im talking about if my explanation wasnt clear enough. If some vital information is missing, I'll provide it down below.
Until then and thanks in advance,
Florian J.
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Hello everyone,
Sorry for late response, it's been some busy two weeks with other more pressing matters on hand. I'll do my best to answer the questions that came up tomorrow when I have access to my data again. (This is just a quick PSA so you dont think I didn't bother to check here again after getting an answer.)
Have a nice sunday and until then
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As we know, an essential oil is a mixture of components playing a pivot role in various domains.
I'm carrying out the antioxidant activity using in vitro tests.
Before doing this task, can we predict the activity of my essential oil only from the compounds found in my sample?
Is there any software to make a theoretical study?
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Can done by molecular docking using computer program but it will take long time
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In order to make a good comparison between your present work and the previous reported studies, we need to compare with results obtained below the same conditions.
Sometimes, we don't find works realised under the conditions that we used !
How can we valorize our work?
Are we allowed to compare even if the conditions are not alike?
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Official definitions by IUPAC
The terms “antioxidant activity” and “antioxidant capacity” have different meanings: antioxidantactivity deals with the kinetics of a reaction between an antioxidant and the prooxidant or radical itreduces or scavenges, whereas antioxidant capacity measures the thermodynamic conversion efficiencyof an oxidant probe upon reaction with an antioxidant.
"Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report)
  • January 2013
  • Pure and Applied Chemistry 85(5)
  • DOI:
  • 10.1351/PAC-REP-12-07
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Recently, FRAP (Ferric Reducing Antioxidant Power Assay) is not accepted by some journals for the investigation of the antioxidant power of a sample like essential oil!
I don't know the reason behind this refusal!
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The performance antioxidant activity of essential oil (EOs) is the result of a complex interplay between components and the oxidizable tested material. ABTS and DPPH are antioxidant assays to measure the activity of the compounds to scavenge free radicals. The essential oil especially terpenoid groups containing phenolic skeleton can react rapidly with peroxyl radical, therefore could protect oxidation by those radicals. Most of the phenolic compounds capable of scavenging free radicals by donating their electrons or H (from a hydroxyl (—OH) in the benzene ring) to the radicals. On the other hand, non-phenolic terpenoids can not protect lipid from oxidation due to (for example alpha-pinene or its similar compounds) undergo auto-oxidation. Furthermore, not all EOs have the ability to reduce reduces Fe3+ to Fe2+ (as measured in FRAP assay), even the phenolic structure with ortho-dihydroxy moiety (ex. hydroxytyrosol) could chelate Fe2+ whereas the tyrasol that has only one OH does not show that ability.
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I'm studying the mobility of amylodogenic tau aggregates in HEK293A cell by using the technique FRAP/FLIP. When I knocked down my gene of interest, FRAP results showed higher recovery rate but that of FLIP didn't change significantly. I believe that FRAP and FLIP are complementary to each other and their results are usually consistent. I don't know how to interpret the inconsistent results of my FRAP/FLIP experiment.
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Hi! First, you should know the photophysics of you dye. For example, may be in FRAP your observed the fast reverse photobleacing recovery.
Second, experimental geometry. FLIP is more sensitive to border proximity. May be the observed fluorecence decrease (logarithmic, in first approximation) strongly interferes with total dye decrease in all area. In FRAP, it will result to appearance of so called immobile fraction, but smaller effect on recovery rate
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There are various methods to measure the antioxidant activity of a compound or sample test, for example, DDPH radical scavenging activity, ABTS assay, TEAC, TRAP, FRAP assay, Superoxide radical scavenging activity (SOD), Hydroxyl radical scavenging activity, Hydroxyl radical averting capacity (HORAC) method, Oxygen radical absorbance capacity (ORAC) method, Reducing power method (RP), etc. What in vitro antioxidant method is the most relevant to biological antioxidant activity (in vivo)?
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Dear Harto Widodo,
please see in
Best regards,
I. Popov
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Hi everybody,
To evaluate the antioxidant activity of a plant, I realised FRAP technique !! In the protocol, there is a centrifugation step, but, once this is end, i didn't see any separation !! Is that normal ? if anyone has already do it, can he help me please ??
Thank's a lot
YB
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Thank you so much Yasmine for your correction. Go a head.
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Hi
I have tried to measure the mobility of a protein of interest by the FRAP assay. The protein was fused with GFP. I overexpressed the fusion protein in Hela cells. However, when the area of the protein was totally photobleached, it can not be recovered even over a long time. If the area of the protein was partially photobleached, it recovered soon. So, is the fluorescent protein must be totally photobleached in FRAP assay? or, it can be partially photobleached? Because I note that in many research papers, it is partially photobleached when the FRAP assay is conducted.
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Dear Chong!
Please You look at following publication:
Figure 1-3
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Specifically am asking for antioxidant analysis( TAC, TFC and TPC) and free radical scavenging activity assays ( ADDH, FRAP AND BTS). it would be great if the reason could also be explained?
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Hi Kajanthan,
The blank in any antioxidant assay must contain all the chemicals involved in the reaction except the plant extract or the standard molecule (antioxidant).
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Hi all,
Im performing FRAP experiments on markers of DNA damage response transiently transfected into the cells. I would like to generate DBSs in a region of interest with 405nm laser and measure the kinetics of the targets there.
I tried to irradiate cells without sensitize them but from IF for gH2AX there was a small amount of damage, even with the highest settings, and the accumulation did not happen at all.
I could use BrDU, but I am not sure if it is suitable for 405 nm laser, any suggestion?
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You are wellcome Paolo Fagherazzi , I hope it helps you with your research. Good luck!
Best!
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I'm using FeSO4.7H2O as my standard. I'm a bit confused what unit i should use. I found some papers used uM(Fe(II)/g dry mass). My unit for different concentration is ug/ml. Should my unit be eg. 50ug/ml (Fe(II)/g dry mass)?
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We used μmol Fe2+.g-1 TP while observing FRAP in semen plasma.
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I have measured the frap using nRhDHPE and NBD DPPE dye before and after treatment with virulent LAM lipids in THP1 macrophages. I got very confusing results even after doing four independent sets. I have observed that there is no change in the diffusion coefficient whereas, the mobile fraction was increased in the raft region and decreases in the non-raft region.
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As LAM was found to disrupt microdomains in artificial membrane (doi: 10.1529/biophysj.107.104075) and potentially enriched in cell membrane nanodomains (doi: 10.1128/IAI.01549-07), a guess would be that if domains are smaller, disrupted, or even in fewer number, you would have more "rigid" lipids from rafts such as NBD-DPPE now in "fluid" membranes. So you would see an increase of mobile fraction from NBD-DPPE, and a decrease with nRhDHPE due to an enrichment of "rigid" lipids in fluid membranes. You might want to try with cholesterol probes
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Dear colleagues, I have evaluated some of my samples for antioxidant activity FRAP, and I determined the EC50 of my samples, and while writing my paper I discovered that some interesting articles had results expressed as mM Fe2+/g, I would like to know if there is any way to convert mM Fe2+/g to EC50 expressed by µg/ml, so I would be able to compare my results to the previously obtained ones, thank you very much.
best regards.
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Do the conversion as follows: 1 microgramme per mL X 1 mMole per 55.84g (molecular weight of Fe2+)=0.0179, appropriately 0.018 micromole of Fe2+ is equaled to 1 microgramme per mL of the same Fe2+. So you can use the conversion factor to convert all your EC50 in vitro values of the results.. I wish you the best of luck!
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I am using Ferric sulphate as standard and ascorbic acid as positive control
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Total Antioxidant Determination by FRAP Method
FRAP assay was carried out according to the previous study with some modification.[16] FRAP reagent was prepared from acetate buffer (1.6 g sodium acetate and 8 mL acetic acid makeup to 500 mL; (pH 3.6), 10 mM TPTZ solution in 40 mM HCL and 20 mM iron (III) chloride solution in proportion of 10:1:1(v/v), respectively. The FRAP reagent was prepared fresh daily and was warmed to 37°C in oven prior to use. A total of 200 μL of samples extract were added to 4 mL of the FRAP reagent and mixed well. The absorbance was measured at 593 nm using UV-VIS spectrophotometer (UV-VIS specord 205). Samples were measured in three replicates. Standard curve of ascorbic acid (125, 250, 500, 750, and 1000 μmol) and GA were prepared using a similar procedure.
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The MRS and YPD both the media are showing antioxidant activity almost the same as probiotic culture supernatant. Is there any method so that I can avoid this issue? Or Can I make a sterile suspension of cells in distilled water and keep it for some hours and use that?
I have done three assays,
ABTS, DPPH and FRAP
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like
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We will be conducting research on colored rice--red, brown, black and white rice and utilize FRAP, superoxide assay and hydroxyl scavenging assay to determine antioxidant activity and correlate it with anti-alphaglucosidase activity in mice. We want to know which Philippine rice variety is best to use and what scientific names are involved in this? Thank you.
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follow
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I am performing a FRAP experiment on honey and I wanted to determine if what is the most suitable incubation time in performing it. As for honey, the incubation time until the plateauing activity can be observed after an hour but I am not sure if whether this is accurate or reliable since reagents tend to degrate over a period of time.
pls help
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I prepared the FRAP reagent using a 10:1:1 ratio of 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 10 mM FeCl3.6H2O.
Why is there a need to incubate the said solution at 37 degrees before adding it to the test samples as well as during the assay proper?
Thanks
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Thank you very much! do you have any references for the said statements?
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After getting the absorbance, how can I get IC50? Should the graph be linear or logarithm graph?
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Hello everyone, You know what is the blank for the FRAP Assay?, to calibrate to zero the FRAP solution, or What is the absorbance of FRAP solution?, (control absorbance, for example in DPPH is around 0.6).
Thank you for the answers
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Hello Manju Tembhre, thanks for the answer, but can you tell me what is the blank to read the absorbance only for the FRAP solution?, without the analyte, because I am working with a methodology that requires the absorbance of only the initial reagents (FRAP solution). Get my kind regards.
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Hello everyone, can you explain me how you do the dilution of the samples (extracts) in the ABTS and FRAP assay?
What solvent do you use and what is the blank for each assay to read the absorbance at 734 and 593 nm.
Thank you for the information
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What is the nature of extract.(i.e. Polar or non polar).
If polar you can dilute the samples in deionized water and also prepare Stock ABTS and pottasium per sulphate in same water.
If sample mid polar, after preparation of stock reagents u dissolved in ethanol or acetone.
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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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Hi Im currently studying the antioxidant activity of Aqueous crude extract of the leaves of Avocado, Papaya, and Mango I don't know what standard to use. I will be utilizing DPPH, FRAP, Hydrogen peroxide and Reducing power assay. Thank you!
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Ascorbic acid is a standard antioxidant and soluble in water.
You can go ahead with AA
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Does anyone know how to calculate IC50 for FRAP?
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Adam B Shapiro agree with
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1- Hydroxyl Radical Antioxidant Capacity (HORAC).
2- Trolox Equivalent Antioxiadant Capacity (TEAC).
3- Total Radical-Trapping Antioxidant Parameter (TRAP).
4- The DPPH.
5-Total Oxyradical Scavenging Capacity (TOSC).
6- Peroxyl Radical Scavenging Capacity (PSC).
7- Ferric Reducing/Antioxidant Power (FRAP).
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Why do you want to measure antioxidan tpropoerties (using the usual colorimetric tests I suppose), it is just not relevant.
Many intervention trials showed that pure antioxidants or phenol-rich extracts cannot prevent radical associated diseases (cancer, cardio-vascular, aging …). Considering recent studies, it appears advisable not to interfere with the delicate internal redox homeostasis of the cell by non-physiological exogenous dosages of phenolic antioxidants:
- The use of total antioxidant capacity as surrogate marker for food quality and its effect on health is to be discouraged, Pompella, A. et al., Nutrition (New York, NY, United States) (2014), 30(7-8), 791-793.
- No correlation is found for vegetables between antioxidant capacity and potential benefits in improving antioxidant function in aged rats, Ji, Linlin et al., Journal of Clinical Biochemistry and Nutrition (2014), 54(3), 198-203;
- Free radicals and antioxidants: myths, facts and mysteries; Lone, Abid A. et al., African Journal of Pure and Applied Chemistry (2013), 7(3), 91-113.).
The debate on the advantages of an additional dietary uptake of antioxidants is long-standing: "Although total antioxidant capacity (TAC) may be helpful in comparing different food items, the extrapolation to their contribution of antioxidant defense in vivo and to health issues should be discouraged, with possible exception of the gastrointestinal tract. This is of particular importance because dietary phytochemicals and other small molecules have other nonantioxidant activities."(Total antioxidant capacity: appraisal of a concept, Sies, H. Journal of Nutrition (2007), 137(6), 1493-1495.)
The EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) more recently inclined to a critical position. The current "Guidance for the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health" https://doi.org/10.2903/j.efsa.2018.5136) not even contains the words "phenol" or "ferulic acid".
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How do one clarify and explicitly state a research gap in a paper, this study was done using crude plant extract and phytochemical analyses of the constituents of the extract was done using colorimetric methods (Total polyphenols, total alkaloids, flavanols and flavonols content) and HPLC; as well as antioxidant activity ( ORAC, FRAP, TEAC and DPPH assays). Numerous previous studies have been carried out on this plant but my species is from the Tropics. How do one close the research gap.
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There is no gape the constituents of the plant and the biological activities are differ according to its location even in the same species. The different methods of estimation also affected the amounts determined. Just write your methods and results and compare with others
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Is it correct to asume that a protein-lipid complex moves slower than a protein alone in FRAP experiments?, briefly, I have a membrane channel fused with YFP, the channel has the capacity to bind diacylglycerol (DAG). Would you consider that under a DAG production situation, a WT channel will move slower (or lower halftime recovery) than a mutant channel that can't bind DAG? Thanks a lot in advance for your answers and suggestions.
Javier
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Javier Casas Yes,I think this is more probable than the converse if the DAG-lipid
interaction is favorable and also because of the larger effective mass of the complex.Did you actually observe slower diffusion?
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after freshly preparing of FRAP reagent i add 3 ml of the reagent to 0.1 ml of solution. but when i tried to take the reading in spectrometer the absorbance value kept increasing for about one hour before reaching little stable stage.
what could be the problem???
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Hey, try this method
1. Add 0.9ml of HPLC grade water to 0.1ml of sample solution.
2. Then add 2ml of FRAP reagent to it and mix well.
3. Incubate it for 30mins at 34 degree Celsius
4. Read the Abs. at 593nm
*FRAP reagent prepared by mixing the following solutions accordingly in the ratio of 10:1:1
300mM Acetate Buffer
10mM TPTZ in 40mM HCl
20mM ferric chloride hexahydrate
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I need to estimate antioxidant in lichens methanolic and acetonic extract samples , by using FRAP method, I'm just searching an easy protocol or any paper help me
… Read more
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Dear read the protocol for the kit you are using.
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I'm doing FRAP assay according to Benzie and Strain's protocol using microplate. I'm confused about the dilution of the sample (it is in g)
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Make 10mg/ml stock of your sample and dilute them as per your working concentrations and please see the reference article I think it is useful to you
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Hi,
Well it's been like the N time while i'm treating this activity "FRAP"
my problem was at first at the end of the analysis with Uv spectrophometer the values are in minuce while it shouldn't be
secondly , the color that i have at the end of the reaction is BLEU while it should be yellow to green depending on the concentration of my preparations tubes ?
Knowing that i've used the very known protocol and also i stuck with it and the the second problem remains each time
can you propose for me a solution ?
could it be the Phosphate buffer Solution ?
what do you think ? Any suggestions i'll be please to read them
Thanks in advance
Best regards
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Ok I'll try that I will tell you about the result
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I want to know more about the link between Ferric Reducing Antioxidant Power and Metal Chelating Activity in antioxidants. I need to know the chemical underpinnings behind using these two methods. Related literature, books available or short answer will be appreciated. Thank you very much...
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First, I don't use these assays. Second, what is the obvious answer?
Third, I would not recommend FRAP assay. This test is performed under much lower pH, ~2, than physiological. Fe(II) is a very active PRO-OXIDANT (https://en.wikipedia.org/wiki/Pro-oxidant). Chelating can reduce this activity.. FRAP measures the amount of Fe(III) reduced by antioxidant. Chelating activity is measured by the amount of Fe(II) bound by a standard chelator. In other words, FRAP measured the antioxidant activity, while other the pro-oxidant activity of Fe(II)
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I need to find out the antioxidant activity of oil samples using FRAP method. Because the chemicals used for the preparation of FRAP reagent contains water (eg. FeCl3 in distilled water) , it is not mixing and forming separate layer on adding oil. I used isopropanol for the dilution of oil.
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You can use DMSO to dilute the extracts
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Hello Everyone,
I am planning to use ferrozine assay to measure the chelation ability of plant extracts (containing polyphenols) by reacting FeCl2 (ferrous) with the extract of interest then add ferrozine to react with the remaining Fe+2 in the solution giving a complex that can be measured spectrophotometrically. I found another paper used ferric-ferrozine to measure the antioxidant power (similar to FRAP assay) by reacting ferrozine with FeCl3 (ferric) to form ferric-ferrozine complex then adding the extract to measure its capability to reduce Fe+3 into Fe+2 and ferrous-ferrozine complex can be measured the same way as the first method.
I am confused between the two methods; can anyone guide me through choosing the best one (I want to measure the chelation ability)?
Thank you,
Fatma
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Chelation ability is best showing using Ferrous-ion chelating activity (FICA) assay. I used it during my PhD to test the ferrous chelating activity of seaweed extracts.
I used the method described by Wang et al. (2009). In a 96-well plate 100 µl of each sample extract (1 mg/ml) were mixed with 150 µl distilled water and 5 µl FeCl2 (2 mM). The reaction was initiated by the addition of 5 µl ferrozine (5 mM). Following 10 mins incubation at room temperature the absorbance was measured at 562 nm using a plate reader . The assay positive control contained distilled water and all assay reagents. The colour blank (containing sample extracts and all reagents apart from ferrozine) was used to correct the colour of the seaweed extracts. The % ferrous ion-chelating activity (FICA) was calculated as follows: FICA, % = (1 - ((Abs562nm Seaweed extract - Abs562nm Colour blank) /Abs562nm Control)) × 100
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I am carrying out the FRAP assay on microbially fermented milk supernatants to assess antioxidant potential. However, some of the milk supernatants give rise to turbidity/cloudiness when mixed with the FRAP reagent. Although I may not see the formation of a blue coloured product, the turbidity causes an increase in absorbance value thereby giving a false positive value for antioxidant activity. Has anyone had and issue like this before? If so, what did you do to rectify the issue in order to get a true value for antioxidant potential when compared to the standard curve. Any help would be much appreciated.
Thanks,
Ken
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Olusegun Abayomi Olalere Ahmed Mostafa Thank you for answering. Your input is much appreciated
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I´m analyzing gap junctional communication by FRAP using carboxyfluorescein. Once inside the cell this dye can pass the membrane only via gap junctions.
We are using a progenitor cell line that can differentiate into neural phenotypes and we compare communication between non-differentiated and differentiated cells. Due to the alterations in cell morphology it is quiet complicated to do FRAP with the same ROI size.
Has anyone an idea how to solve this problem? Maybe normalization to the ROI area?
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Can anybody explain me how to calculate antioxidant properties of a crude plant extract as"mg ascorbate per g dry extract” from FRAP assay
The equation for Vit-C is y=0.0084x - 0.1222 (R2=0.9954). Absorbance sample is 0,157 and concentration sample is 200 ppm
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I agree with Dr. @Sabry Abdallah
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I am doing research on FRAP (TPTZ reagent method) for antioxidant activity. I know the preferred wavelength is 593nm. Is it possible to measure at 570nm instead as for now the 593nm filter not available in the lab. I am using microplate reader as my instrument. Thank you.
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Thank you Syed Mukhtar Ahmed, this assay is called FRAP assay (ferric reducing anti-oxidant power).
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I want to do TAC by FRAP, RPA, DPPH and ABTS method. In place of trolox standard, please suggest me anyother standard
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Thanks to all of you.
But one more enquiry, the concentration will be same as for trolox standard or it will be different? and if its different please guide how should i calculate?
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The problem is that my Antioxidant solution is soluble in Chloroform, so how can I perform this Assay as standard protocol is to make solution of Ferric chloride, ferry cyanide, trichloroaceteic acid in distilled water only?
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Antioxidant activity must be evaluated under physiological conditions. This means either in water at neutral pH or in nonpolar organic solvents (mimicking lipids) such as alkanes. There is no sense to measure antioxidant activity of compounds dissolved in chloroform. First, make your extract soluble in water or in heptane/octane
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I'm working with RSM and some doubts have arisen, I'd be very thankful if someone could help me. I'm working with the optimization of polyphenols extraction and analyzing the antioxidant activity (response, dependent variable) by three different methods (ABTS, FRAP and Total Phenolic Content). I would like to know :
1) What does it mean the quadratic effect being significative or not? Which is it influence in the response?
2) What does it mean if all the effects of the variables (linear, quadratic, mean/interc, interaction) are significative? Does it mean that my model is incorrect?
3) I obtained an ideal extraction condition, with a specific temperature and a specific concentration of the extractor solvent. In ANOVA analysis I obtained that the lack of fit isn't significative (p>0,05), so I understand that the model fits well and is predictive. When I insert the values of X and Y variables of the experimental design (DCCR), the predicted and the observed values are very close, the relative error is small. But when I insert the conditions of temperature and concentration of the extractor solvent (that is a condition that is in the interval studed, not a point of the complete design) in the correct terms of the mathematic model (X and Y values), I obtain a predicted response very different from that one observed in the laboratory analysis. The observed value is higher than the predicted.
4) I made an experimental design with alfa value = +/- 1,41 (Q values), I have 5 levels for each independet variable. If the Q values aren't significant in the effects analysis, is it correct to say that my planning is a first order experimental design? Should I repeat all the optimization analysis again but only with the levels +/- 1,00 for each variable?
Thank you for the support!!
Best
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You say you have 5 levels by factor and a value of alpha of 1.41 (square root of 2). So, I suppose you realized a central composite design with 2 factors. Am I wight?
In such a case, during the analysis of results, removing non statistically significant effects and minimizing the PRESS (maximizing the prediction R²) will give you a quite stable model. If your response vary very much (max/min is about 100 or 1000...) it could be interesting to use a transformation of your response (Box-Cox transformation) or model the log of your response or inverse (these 2 last cases are particular cases of Box-Cox transformation).
With data, it would be more simple to answer.
Hope this can help you.
Pierre Chagnon
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I hav taken absorbance of FESO4  at range of 2.5-200 uM which show linearity with R2=O,98...then i took absorbance of standard/extract at different conc .....how to get FRAP activity...show me calculation?.
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Hi Dr Chibuike Udenigwe,
Can you help me for the calculation part. I am stucked..
For example, my sample has a FRAP value og 1.53 mM FE.. Then, I want to express it in mM Fe/g DW.. So how?
Thank you.
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I have been working on antioxidant activity using FRAP assay. However, the calculation for FRAP assay is 
FRAP value of Sample (μM)
= (Change in absorbance of sample from 0 to 4 minute / Change in absorbance of standard
from 0 to 4 minute) X FRAP value of standard (1000 μM). I am confused with regard to Change in absorbance of standard from 0 to 4 minute. What should be the concentration of the standard with regard to the change in absorbance of standard from 0 to 4 minute?
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First of all, you should choose the way in which you are going to express your results.
For example, you could measure the absorbance change of a standard antioxidant sush us trolox, gallic acid, ascorbic acid or else you can use the IC50 value.
In my opinion, make a literature review about frap calculation in the sample you work on. There are plenty of published works.
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In the context of the Antioxidant activity, based on different activities such as DPPH, NO, and FRAP etc ... different reactions were involved. When exactly radical quenching will take place and when radical scavenging will take place. What is the difference?
Can someone pls clarify my doubt.
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Free radical is defined by the occurrence of an unpaired electron in an organic molecule or metal. This potentially unstable electronic configuration can react either to acquire or to lose an electron and form a more stable product. Free radicals have the potential to serve as either effective oxidants or reductants while Free radical quenching can be defined as any reaction in which the free radical forms products that are not free radicals.
I hope this information is helpful.
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There are several methods explained. So i am bit confused.
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Plz, don't spam the RG forum. Combine your three similar questions about ABTS, DPPH, and FRAP assays into a single question.
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Most of the time there is variation in the performance of this extract relative to the standard eg. ascorbic acid from one assay to the other.
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Antioxidant action for different assays (regardless of mechanism) is similar in the sense of quenching/reducing reactive species/radicals or oxidant probes, but kinetics, potential for side reactions, and dependence on reaction conditions may differ.
no single “universally accepted” assay is adequate in itself to precisely and quantitatively detect/determine all actions of a putative antioxidant.
For more details, see the links below
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I want to determine the antioxidant capacity of lemongrass leaves using the different spectrophotometric assays (ABTS, DPPH, and FRAP) and find its correlation with the total phenolic content. Another aspect I'm interested in involves comparison of the performance of these assays in order to determine which would be the most appropriate to use for my sample. I have been advised to validate the procedures using these parameters: recovery, repeatability, limit of detection, limit of quantification, and linearity. However, I have become confused on how to validate the methods exactly. Can someone please advise on how I should go about with my procedure? How can I determine/calculate these parameters? I'm planning to use Trolox for the standard. Any form of input would be highly appreciated. Thank you very much.
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Noted. Thank you all for your valuable input.
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I have done FRAp assay for Purselaneusing FESO4 as a standard.how do i calculate the values
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Dear Sir. Concerning your issue about the calculation of FRAP value of plant extract. The ability to reduce ferric ions was measured using the method described by Benzie and Strain. The FRAP reagent was generated by mixing 300 mM sodium acetate buffer (pH 3.6), 10.0 mM (tripyridyl triazine) TPTZ solution and 20.0 mM FeCl3.6H2O solution in a ratio of 10:1:1 in volume. Samples at different concentrations (100,200,300,400 and 500 μg/ml) was then added to 3 ml of FRAP reagent and the reaction mixture was incubated at 37 °C for 30 min. The increase in absorbance at 593 nm was measured. Fresh working solutions of FeSO4 were used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mmol FeSO4 equivalents per gram of sample (DW). I think the following below links and the attached file may help you in your analysis:
Thanks
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In my FRAP assay I am expressing my sample's antioxidant capacity in trolox equivalent, TE mg/g. I have got a good standard curve with R2= 0.9997 and samples of different concentrations have given good proportional absorbance values as of standard. But after calculating I have got TE mg/g values which decrease with increase of concentrations. I have validated my calculations in various ways and confident with the calculations. In my general concept, it is more logical to me that higher concentrated sample should show more TE mg/g value. Then why I am getting opposite result repeatedly? Please share your valuable ideas. Thanks.
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Dear Nazma
If the slope of absorbance vs concentration graph of standard is also positive as that of sample then there may be chance of error in reagent preparation or in method. Take care in reagent preparation and procedure 
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FRAP ASSAY
Assay: Experiments were done according to [Benzie I.F., Strain J.J.: The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem., 1996; 239: 70-76] with modifications. FRAP working solution was prepared freshly each time: 0.3 M acetate buffer (pH=3.6), 0.01 M TPTZ (2,4,6-tripyridyl-s-triazine) in 0.04 M HCl and 0.02 M FeCl3 6H2O were mixed in 10:1:1 (v/v/v) and kept away from light. 0.075 ml of phenols (final concentration 0.0001-0.01 mg/ml) or FeSO4 7H2O (final concentration 0-1.8 µmol Fe2+/ml) solution were added to 2.25 ml FRAP working solution and 0.225 ml of deionized water. The mixture was vortexed and incubated at 37°C for 30 min
away from light. Absorbance was measured at 593 nm using the spectrophotometer. FRAP working solution with deionized water instead of a sample was used as a blank.
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I'm using the fluorophore Nile Red to image what happens to lipid samples over time in solution. Everywhere you look people talk about photobleaching, but in my case the sample gets brighter over time (e.g. after 1 hour of non-stop imaging).
The same thing happens with FRAP analysis; the bleached spot brightens after recovery.
Can anybody tell me what might be happening?
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Johannes, as Peter mentioned , please give sufficient time for incorporation of NR in hydrophobic lipid domain and then performed the experiment.
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hello someone who is knowledgeable about some work done antioxidants stability of an encapsulated orange pulp which has assessed the methods FRAP, ABTS, DPPH, and TOTAL PHENOLS.
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Different literatures read spectrophotometer in different time, I am very confused how and when to read the numbers of spectrophotometer and how to zero it. Please let me know if you can clarify this to me. many thanks in advance
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Perv, I always measure at 593nm. I blank/ zero it using FRAP reagent+water.
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I am working on antioxidant properties of orchid species.
For that i did DPPH, FRAP assay. Now want to carry out ABTS assay.
I have reffered many research papers. In some, trolox used as a standard  where as in some Gallic acid used as standard for ABTS assay.
Which one is Best or both can be used as a standard?
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I would choose the standard depending on the matrix: re human nutrition (food, beverages, blood, bile, ...) it would be the best to use Trolox (vitamin E derivative), for antioxidants in plants per se I would use gallic acid.
If money is an object, than use gallic acid for all analyses.
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I used to carried out FRAP experiments also on COS7 cells on filopodia and it worked, the bleaching and the recovery was as expected. Now we would like to try the same on COS7 cells overexpressing an other protein and perform FRAP on the cell body and in the nucleus, but I cannot see any changes in the fluorescence intensity before and after the bleaching on the cell body. I can think about because of the thickness of the cell body, lasers cannot bleach the region of interest. May be the recovery would be such a fast mehcanism, but I have doubts about this one.
Is there anybody who has experiencies using FRAP within the cell body or the nucleus?
Thank you for your answers in advance!
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dear elek,
just try it out and play with the parameter - and then think also what it means to change the parameter, you know you should have a result in the end which is independent of certain parameters, or better you should always have an effect, clearer or not so clear, but if you get only a result because of a very special setting, then the question is: do you measure a real effect or just an effect because of...
consequently, you should have a logical explanation why a certain parameter set works better or worse than another one...
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The literature on this is confusing. I know the original paper did 0 min and tested every 15s for 8 min but we do not have an automated system. What is the best possible way to combat this situation? Thank you!
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Thank you!
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Hi guys,
I just would like to know if you could recommend me any good laser company for purchasing for this CW laser I am interested in. 
I would be very grateful if by your experience you know of a good one you could recommend me. 
Many thanks,
Yurema
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If  50mW , nearly perfect TEM00, all solidstate, CW at 473 nm is enough, I would recommend http://www.spectra-physics.com/products/cw-lasers/excelsior#specs
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I want to determine FRAP of Protein hydrolysate from Acetes
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Udenigwe is correct. Have a look at the attached protocol for details.
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is it possible to use DPPH and FRAP methods to asses the total antioxidant capacity in rat semen? and what is better, to use one of these methods or the total antioxidant capacity kit (TAC assay kit)?
Thank you.
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you are welcome 
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In FRAP, how do we decide the wavelength of emission filter to be used? We are presently using a LASER of 488nm, Dichroic mirror (DM) of 510nm and emission filter of 520nm. Can we use an emission filter of 505nm and how it is going to affect the counts we obtain?
Secondly, if we remove the emission filter from the path we observe some leakage of LASER light and a bluish tinge is observed along with the fluorescence signal even if DM is present. Is it essential to have emission filter in path in addition to DM?
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You can use the 505nm emission filter if it is long pass filter, or if it is a band pass filter extending beyond 510nm, as this is your dichroic cut-off. How it affects the counts you obtain will depend on what the two filter specs are (the 520 and 505), and where the emission maximum of your sample lies.
Whether it is essential to have an emission filter as well as the dichroic depends on how high contrast the sample you are looking at is, but since dichroics are often only 90% efficient, it is usually a good idea to use an emission filter.
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I'm new with FRAP assay. when I mixed FeCl3 with buffer solution befor the addition of sample, a light blue color is formed. Is that a problem?? or its normal?
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hello Mr. Palani, i know what do you describe and it is a problem! First, ensure that the acetic buffer is on room temperature. Then put 1 ml of TPTZ solution in 10 ml buffer and then add 1 ml of FeCl solution. This working solution takes a straw-brown color in 37o C.
i had this blue-purple color when my acetic buffer was cold or had been made time ago.
Good luck!
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I would like to study cell coupling via gap junctions in cultivated cardiomyocytes by using FRAP. I already anaylsed dye spreading with lucifer yellow and found increased coupling. To verify this experiment I thought about FRAP. But I do not know which is the best fluorophore for FRAP? Calcein? Any other suggestions? Thank you!
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Thank you!
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It can be seen in several in vitro antioxidant assays that alpha-tocopherol is used as positive control. But I can not find any paper in which this compound has been used as positive control in FRAP assay.
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I use tocopherol in the test of  phenols,,catechins and vitamins.
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Food chemistry, Pharmaceutical chemistry
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Both of them are spectrophotometric methods, which are used to investigate the in vitro antioxidant capacity of foods and biological samples. They are based on electron transfer reactions, which visually results on the reduction of a coloured oxidant (DPPH or FRAP as oxidant). Therefore, usually the results obtained from these methods present excellent correlation.
I believe the main difference is that DPPH is a stable organic nitrogen radical. The reaction progress between the radical and the antioxidant is monitored for aproximatelly 30-40 minutes at 515-517 nm, or until stability. So, we must be careful with antioxidant compouns such as some anthocyanins that absorb at similar wavelenghts and could interfere in the final results.
In the case of FRAP, there are no free radicals. What is monitored is the reduction of ferric iron to ferrous iron in the presence of the antioxidant. This is usually done every 15s until 4 minutes, at 597nm. For some polyphenols, FRAP values cannot be accurately obtained after this time.
The file article attached (The chemistry behind antioxidant capacity assays.) may be helpful.
Hope it could be helpful.
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I need to calculate the scavenging activity , and what is the correlation between DPPH assay and FRAP assay?
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The mechanism of FRAP assay and DPPH scavenging assay is similar i.e., these methods measure the electron transfer ability of antioxidants. Therefore, higher correlation has been reported by many researchers. Moreover, FRAP assay gives excellent result for polyphenol rich compounds (antioxidants). It is better to use at least one alternate method based on hydrogen atom transfer ability. You can select either Oxygen Radical Absorbance Capacity (ORAC) or Low-Density Lipoprotein (LDL) Oxidation or Total Radical-Trapping Antioxidant Parameter (TRAP) or other assay methods.   
For methodology of FRAP assay please go through link below
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i have prepare linear curve of 2.5micrmolar FeSO4 to 20 micrmolar FeSO4...also i have taken absorbance of standard Ascorbic acid and my extract at different concentration ....Then how to calculate FRAP activity?
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Dear Vikas,
I have copied the experimental section of FRAP assay from a publication entitled " ANTIOXIDANT ACTIVITY OF ETHANOLIC EXTRACT OF Maranta arundinacea .L TUBEROUS RHIZOMES" Published in Asian Journal of Pharmaceutical and Clinical Research Vol 5, Issue 4, 2012 (for full paper see attached file) for quick view. You should make a curve such as that illustrated in Figure 6 of the paper.
THERE IS NO CALCULATIONS TO BE DONE. ONLY TO SHOW GRAPHICALLY THE EFFECT COMPARED TO STANDARD.
FRAP Assay
Frap assay measures the reducing potential of an antioxidant reacting with a ferric tripyridyltriazine [Fe3+-TPTZ] complex and producing a coloured ferrous tripyridyltriazine [Fe2+-TPTZ]11. Generally, the reducing properties are associated with the presence of compounds which exert their action by breaking the free radical chain by donating a hydrogen atom26.Frap assay treats the antioxidants in the sample as a reductant in a redox-linked colorimetric reaction27. In the present study, the trend for ferric ion reducing activities of M.arundinacea and BHT are shown in Fig 6. The absorbance of M.arundinacea clearly increased, due to the
formation of the Fe2+-TPTZ complex with increasing concentration. The water and ethanol extracts of sumac (Rhus.coriaria L.) showed increased ferric reducing power with the increased concentration as standard antioxidants28. Hence they should be able to donate electrons to free radicals stable in the actual biological and food system.
Fig 6:FRAP Assay
The ethanolic extract of M.arundinacea was found to be an effective scavenger of ABTS, DPPH, H2O2, and NO and also possessed a good reducing power and Frap activity. Earlier reports on the antioxidant activity of M.arundinacea are very rare in the literature. Therefore, it is very difficult to compare our results with that of previous studies. The high antioxidant activity of M.arundinaacea enhanced the potential interest in these under-exploited tubers for improving the efficacy of different products as nutraceutical and pharmacological agents. The consumption of the arrowroot may play a role in preventing human diseases in which free radicals are involved, such as cancer, cardiovascular disease, and aging. We conclude that, the results presented indicate that M.arundinacea extract attenuated oxidative stress via its antioxidant properties. However, further investigations on phenols,  flavonoids, active principle, their in vivo antioxidant activity, and the different
antioxidant mechanism are warranted.
Hoping this will be helpful,
Rafik
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I have extracted plant samples using four different solvents. I found that the DPPH scavenging activity of all tested samples are averagely similar but the FRAP reducing result of a particular sample seems to be very much higher than the rest of the samples. What does this signify? How can I interpret this result?
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Colour controls are OK, but I guess the question is how to discriminate whether or not the observation of high FRAP activity in the sample is real,  or there is an artefact. As redox reactions are involved, we should be sure there is not pH change among the assays of the different sanples when FRAP reagents is used.
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I ve got the negative equation for my linear regression in FRAP standard curve. most of the paper they get positive equation. I wonder how i get negative and may i just use that negative equation for my calculation? thank you.
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what equation did you get?
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I have absorbance values for my extracts when I tried FRAP assay but I need the results in units and not just absorbance.  Can anyone help urgently? 
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Hi Ashlesha. You will need to do a standard curve using FeSO4 and also use a standard concentration of ascorbic acid. Have a look at my attached FRAP protocol. if you need help just ask.
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Hello,
I have to perform FRAP experiments using fluorescent labeled cholesterol tBLM. According to lateral diffusion properties, which fluorophore is more indicated for photobleaching? We have NBD and Cy-5.
Thanks,
Giulio 
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A highly photobleacheable fluorophore with a high brightness... This would be the best obviously!
I would go for NBD, not because of its photophysics properties, but because of its chemical structure... I expect it to less perturbates the BLM than the Cy5 will.
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I have seen studies of the diffusion and exocytosis of a neurotransmitter receptor in organotypic hippocampal slices done by FRAP but can you study it at some extent with a biotinylation assay (labeling intracellular and surface- labeled proteins)?
I don't know much of these techniques. What would you reckon is the major drawback of these techniques?
Many thanks in advance!
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i did not check, but biotin label is not good for live study. Simple you do not know, what you see... The label is not specific. If specificity is not an issue,  you can use it, i think.
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I have taken a 40 mg of sample in 3 ml of methanol and used 7.5 microlitre of sample in my assay. Please guide me in the back calculation to convert O.D in micromolar /g of dry weight?
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You have to prepare a calibration curve of the selected standar in micromolar (X axis) and O.D (Y axis). With the linear equation you can interpolate your O.D. results. Then you have an amount of 0.1 mg of sample in the 7.5 uL of analysis (0.0001 g). So, if you divide the micromolar obtained in the 0.0001 g of sample you get your results in micromolar/g. Now it depends if you need to express in grams of extract or grams of plant/frui/ etc, you have to relate this result with the yield of extraction.
I hope this can be helpful. Please find attached a publication were we used the FRAP assay for the antioxidant activity of a fruit extract.
Best regards, 
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We are investigating the anti-oxidant properties of herbs using ABTS assay, I saw in a different paper that they use FRAP for the algae. Can I use ABTS assay for the algae?
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Dear Bernard,