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  1. Link

    total-antioxidant-capacity-assay-kit-ab65329.pdf

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Total Antioxidant Capacity Assay Kit (ab65329)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (3)Q&A (34)References (54)

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Functional studies - ab65329
  • Functional assays: TAC (ab65329)
  • Functional assays: TAC (ab65329)
  • Functional assays: TAC (ab65329)
  • Typical Trolox standard calibration curve

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell culture media, Cell Lysate, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine

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Overview

  • Product name

    Total Antioxidant Capacity Assay Kit
  • Detection method

    Colorimetric
  • Sample type

    Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate, Cell culture media
  • Assay type

    Quantitative
  • Assay time

    2h 00m
  • Product overview

    Total Antioxidant Capacity Assay Kit ab65329 can measure either the combination of both small molecule antioxidants and proteins, or small molecules alone in the presence of our proprietary Protein Mask.


    In the total antioxidant capacity assay protocol, the Cu2+ ion is converted to Cu+ by both small molecule and protein antioxidants. The Protein Mask prevents Cu2+ reduction by proteins, enabling the analysis of only the small molecule antioxidants. The reduced Cu+ ion is chelated with a colorimetric probe giving a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity.


    Total antioxidant capacity assay protocol summary:
    - add protein mask to samples if only measuring small molecule total antioxidant capacity
    - add samples and standards to wells
    - add Cu2+ solution and incubate for 90 min at room temp
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K274 Total Antioxidant Capacity (TAC) Colorimetric Assay Kit. K274-100 is the same size as the 100 test size of ab65329.

    Antioxidants play an important role in preventing the formation of, and scavenging of, free radicals and other oxidizing species. There are three categories of antioxidant species: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbate, uric acid, GSH, vitamin E, etc.) and proteins (albumin, transferrin, etc.). 

    Trolox is used to standardize antioxidants, with all other antioxidants being measured in Trolox equivalents. Measurement of the combined non-enzymatic antioxidant capacity of biological fluids and other samples provides an indication of the overall capability to counteract reactive oxygen species (ROS), resist oxidative damage and combat oxidative stress-related diseases.

    Related products

    Review the oxidative stress marker and assay guide to learn about more assays for oxidative stress.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components Identifier 100 tests
    Assay Diluent WM 1 x 10ml
    Cu++ Reagent Blue 1 x 0.2ml
    Protein Mask NM 1 x 10ml
    Trolox Standard (1 µmol) (Lyophilized) Yellow 1 vial
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Cell Damage Kits
    • Oxidative stress
    • Kits/ Lysates/ Other
    • Kits
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    • Cell Damage
    • Kits/ Lysates/ Other
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    • Oxidative Stress Assay Kits
    • Oxidative Stress
    • Kits/ Lysates/ Other
    • Kits
    • Cell Damage Kits
    • Antioxidant
  • Relevance

    Antioxidants play an important role in preventing the formation of and scavenging of free radicals and other potentially toxic oxidizing species. There are three categories of antioxidant species: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbate, uric acid, GSH, vitamin E, etc.) and proteins (albumin, transferrin, etc.). Different antioxidants vary in their reducing power, and cooperation of all different antioxidants provides greater protection against reactive oxigen or nitrogen radicals than any single compound alone. Therefore, the overall total antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual components, as it considers the cumulative effect of all antioxidants present.

Images

  • Functional studies - ab65329
    Functional studies - ab65329Image from Wan X et al., PLoS Pathog 12(10), Fig 5D. doi: 10.1371/journal.ppat.1005954 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Total antioxidant capacity was determined in mouse cardiac homogenates using Total antioxidant capacity assay kit (ab65329). 

  • Functional assays: TAC (ab65329)
    Functional assays: TAC (ab65329)

    Trolox equivalent capacity measured in milk and concentrated squash. Background signal subtracted, duplicates; +/- SD

  • Functional assays: TAC (ab65329)
    Functional assays: TAC (ab65329)

    Trolox equivalent capacity measured in mouse tissue lysates, showing quantity (nmol) per mg of extracted protein. Results following blocking of protein activity is shown (Mask). (Duplicates; +/- SD).

  • Functional assays: TAC (ab65329)
    Functional assays: TAC (ab65329)

    Trolox equivalent capacity measured in biological fluids. Results following blocking of protein activity is shown (Mask). Background signal subtracted, duplicates; +/- SD.

  • Typical Trolox standard calibration curve
    Typical Trolox standard calibration curve

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (54)

Publishing research using ab65329? Please let us know so that we can cite the reference in this datasheet.

ab65329 has been referenced in 54 publications.

  • Madariaga VI  et al. Myogenous temporomandibular disorders and salivary markers of oxidative stress-A cross-sectional study. J Oral Rehabil 48:1-9 (2021). PubMed: 32979853
  • Li M  et al. Brachyury engineers cardiac repair competent stem cells. Stem Cells Transl Med 10:385-397 (2021). PubMed: 33098750
  • Okda TM  et al. Chemopreventive and anticancer activities of indomethacin and vitamin D combination on colorectal cancer induced by 1,2-dimethylhydrazine in rats. Biomed Rep 14:27 (2021). PubMed: 33408861
  • Shili CN  et al. Effect of a Phytogenic Water Additive on Growth Performance, Blood Metabolites and Gene Expression of Amino Acid Transporters in Nursery Pigs Fed with Low-Protein/High-Carbohydrate Diets. Animals (Basel) 11:N/A (2021). PubMed: 33672517
  • D'Onofrio N  et al. Phenolic Profiles of Red Wine Relate to Vascular Endothelial Benefits Mediated by SIRT1 and SIRT6. Int J Mol Sci 22:N/A (2021). PubMed: 34073604
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

11-20 of 37 Abreviews or Q&A

Question

Can the kit measure the antioxidant activity of an extract of a plant? If Yes, may i know the method to measure... waiting for your reply... with thanks.

Read More

Abcam community

Verified customer

Asked on Feb 26 2013

Answer

This kit has only been optimized for mammalian sample. Theoretically, it could be used in samples of multiple species including plant. Because we have not tested it with any plant sample, I am sorry that there is no specific protocol for measuring plant sample.

In order to get the most efficient result, you may have to optimize the original protocol.

Original protocol for preparation of tissue sample:

For Tissue Samples, Start with 10-100 mg of the tissue, add 500-1000 μl (or ˜4-6 volumes) of the PBS on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Sonication can also be performed instead. Spin down the sample at 10000 rpm for 10 min at 4°C and collect the supernatant. Use the supernatant for the subsequent assays.

Read More

Abcam Scientific Support

Answered on Feb 26 2013

Question


Inquiry: Dear sir/madam I'm going to measure total antioxidant capacity intracellular mouse spermatozoa, Is kit ab65329 appropriate for this measurment? if your answer is YES, how many mouse spermatozoa is needed for this measurment? if your answer is NO, do you have any kit for this measurment? I'm looking forward to hearing from you as soon as possible. Best regards

Read More

Abcam community

Verified customer

Asked on Dec 07 2012

Answer

Thank you for your enquiry.


I am sorry to confirm we have not specifically tested mouse spermatozoa using this kit. But since the kit is compatible with cells of murine origin, I do not see why it would not work.

We would recommend to start with a relatively large number of cells, ˜ 2 - 5 x 10^7 cells. Lyse them in the assay diluent and use different amounts for the assay to find the optimum.

Hope this information has been useful for you. Please do not hesitate to let me know if you have any other questions.

Read More

Abcam Scientific Support

Answered on Dec 07 2012

Question

Dears,

Does this kit works well on Saliva?


Total Antioxidant Capacity Assay Kit (ab65329)





Kindest regards,

Read More

Abcam community

Verified customer

Asked on Nov 13 2012

Answer

Thank you for your enquiry regarding ab65329.

As the datasheet indicates different biological fluids can be used with this assay kit. Although we have not used ab65329 with saliva samples - this type of same would be compatible. I would recommend treating it the same way as any other liquid samples (serum/plasma) and follow the protocol exactly.

If you need any further assistance in the future, please do not hesitate to contact me.

Read More

Abcam Scientific Support

Answered on Nov 13 2012

Question

dear sir
I ask about the componenents what do you mean by abbrevation( DMSO) in standard trolox?

also ask about which type of plot standard curve use (lineer regration,ect........? or slope of standard curve

If this kit measure all or total antioxidant capacity in saliva , and way or protocol for measuring in saliva sample?

and perpration of standard curve?

please supply complet mthdology about using kit

Read More

Abcam community

Verified customer

Asked on Nov 12 2012

Answer

Thank you for contacting us.

With regards to your questions:

1. DMSO is a solvent that dissolves both polar and nonpolar compounds. In this case DMSO is used to dissolve lyophilized Trolox standard.

2. The type of standard curve for this kit is a linear regression.

3. We have not tested saliva with this kit so I am sorry we are unable to provide the optimized sample preparation guide. The optimization should be determined by the end user. I would also suggest consulting literature for example;

http://www.ncbi.nlm.nih.gov/pubmed/11940135

http://www.ibra.org.uk/downloads/20120109_33/download

http://www.ptfarm.pl/pub/File/Acta_Poloniae/2009/5/477-482.pdf

As a guideline you can follow the following procedure.

1. Collect saliva in a clean beaker or flask and store on ice.
2. Optimize the dilutions by serial dilution or as required.
3. For long term storage saliva samples can be stored at -80C.

I am attaching the protocol booklet which has the instructions about how to use this kit ans how to prepare a standard curve. Briefly, to prepare a standard curve:

Add 0, 4, 8, 12, 16, 20 μl of the Trolox standard to individual wells.
Adjust the total volume to 100 μl with ddH2O to give 0, 4, 8, 12, 16, 20 nmol of Trolox standard.
Read the absorbance at 570 nm using the plate reader.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Abcam Scientific Support

Answered on Nov 12 2012

Question

I would like to order Abcam TAC kit (ab65329), but there is no information about tissue homogenization process, can be used to measure the total antioxidant capacity of rat liver tissue homogenate and if so how to prepare the sample for the tissue homogenate? Which % buffer do I need to use? spin of centrifuge, how many rpm or g? and how long? can i use sonicator for homogenization?

Read More

Abcam community

Verified customer

Asked on Oct 12 2012

Answer

Thank you for contacting us. Yes, this kit will be suitable for use on rat liver samples.

Here is the protocol for tissue homogenization for this kit: Start with 10-100 mg of the tissue, add 500-1000 ul (or ˜4-6 volumes) of PBS on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed by viewing the cells under the microscope. Alternatively, sonication can be performed. Spin down the sample at 10,000 rpm for 10 min at 4C and collect the supernatant. Use the supernatant for the subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

I hope this helps, please let me know if you need any additional information or assistance.

Read More

Abcam Scientific Support

Answered on Oct 12 2012

Question

Yes, please send us a replacement kit.

Thanks,

Read More

Abcam community

Verified customer

Asked on Sep 20 2012

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1167932.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Abcam Scientific Support

Answered on Sep 20 2012

Question

The Cu reagent was made fresh each day for each run. Thank you for looking into this issue.

Read More

Abcam community

Verified customer

Asked on Sep 19 2012

Answer

Thank you very much for your patience.

Regrettably we couldn't find any reason for dropping off the standard curve. Our lab has re checked each and every aspect of protocol however they are unable find any quality issues.

On the other hand we have sold many units without receiving any complaint so the quality of the kit is not questionable.

Finally only thing we can suggest is, trying a replacement kit. Let me know if you are interested I will then send you a replacement kit.

Read More

Abcam Scientific Support

Answered on Sep 19 2012

Question

Dear Mr or Ms:

I’m interested in ab65329 Total Antioxidant Capacity Assay Kit.

Question

1,The kit detects both small molecule antioxidants and/or proteins, except for enzymatic antioxidants. Since total antioxidant consists of three parts, how can we say that this kit measure “Total Antioxidant Capacity”?

2,which sample is better between serum and plasma? Why? Which anticoagulant is better respectively?

3,my sample is plasma-EDTA, can it be tested with this kit?

4,The sample has been stored under -80 for a year, can it be tested? How long sample be stored under -80?

5,To draw the standard curve, “Add 0, 4, 8, 12, 16, 20 μl of the Trolox standard to individual wells.Adjust the total volume to 100 μl with ddH2O to give 0, 4, 8, 12, 16,20 nmol of Trolox standard.” it’s not serial dilution. That is ,we first make 32mM solution ,then we dilute 32mM solution to 16mM.then we use 16mM solution to make 8mM solution and so on. Do you think which method is better?

6,Do we need multichannel pipet when we have a quantity of samples?

7,does the kit provide microplate? It should be clear, black or white? flat- or U-shaped

Wells?

8,do we need to avoid the light? In which step?

9,Do the blood samples need to avoid light?

10.We put the reagents and blood sample in roon temperature?Ice?Which reagents?

11,When we do the experiments at different time,we make standard curve and black control every time?When we have more than one plates,we make standard curve and black control on each plate?

12,when we centrifuge the blood to get plasma, there is little remaining red blood cells, does it matter? do we need to centrifuge the plasma again before we test?

13,does it need to triplicate for standard,blank and sample?

14,“The units of measurement will be in nmols/ul or in mM of Trolox (i.e. the equivalent thatyou will the comparing the sample to). ”Cited from your website.But in the publications you provide,the unit is10.12+1.65 (µM), (Effects of Pycnogenol on endothelial function in patients with stable coronary artery disease: a double-blind, randomized, placebo-controlled, cross-over study),

is the result reliable?

Too many questions, thank you very much in advance.

Read More

Abcam community

Verified customer

Asked on Sep 14 2012

Answer

Thank you for contacting us.
1. This kit will measure only the total non-enzymatic antioxidant capacity of the samples.
2. Both human serum and plasma have an antioxidant capacity of 0.5-2mM. So both samples would be equally good for detection.
3. Although I don’t think EDTA will have an adverse effect on the efficiency of this kit, I would not recommend using EDTA for plasma preps. Instead use heparin or citrate.
4. If the samples are stored well at -80°C you can use it within the year.
5. The standards we recommend are not serial dilutions. Please use them as recommended.
6. Yes, a multichannel pipette would definitely help, but it is not an essential prerequisite with this kit.
7. The kit does not come with a plate. Please use a clear flat bottom plate.
8 and 9. This is a colorimetric assay. You don’t need to shield it from light.
10. Store all kit components in unopened form (as they arrive) at 4°C. After reconstitution, the standards need to be stored at -20°C.
11. Every time you do an expt, you will need to make new standards and blanks.
If you are doing 2 plates at the same time, it will be ok if you do standards in just 1 plate as long as you treat both plates exactly the same way.
12. Yes, use clear plasma, not plasma contaminated with red blood cells.
13. Duplicates for standards and blank should be ok.
14. Please go by the units in our protocol. In either case, 1 mM will be equivalent to 10^3 µM. So the units are the same even though they are represented differently. They are in terms of Molarity.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Abcam Scientific Support

Answered on Sep 14 2012

Question

The standard has been stored at -20* until it is needed, everything else has been stored at 4*. All of the blank readings have been between 0.07 - 0.08. The standard curves I have sent were all calculated after blank subtract. I am confident this is not a usual issue with the kit, but currently this problem is inhibiting our analysis. I have been in contact with the people who initially received the package containing the kit, and it was apparently "hot" or at room temperature for a period of time before being received, could it be possible that this is the root of the problems we have been seeing?

Thanks,

Read More

Abcam community

Verified customer

Asked on Aug 28 2012

Answer

Thank you for your enquiry regarding ab65329.

My colleague is out of office and he has asked me to look after her customers during her absence.

For this particular kit - the standard is Trolox which is very stable.

Could you please confirm how the standard was reconstituted and stored? Did you freeze down the whole volume of reconstituted standard solution? How many times you used the standard solution? Was it only in one experiments or in severalindependents experiments?

I look forward to hearing from you and hope to solve this problem as soon as possible.

Read More

Abcam Scientific Support

Answered on Aug 28 2012

Question

Hello,
I have attached the survey for the standard curve issue we seem to be experiencing, I have tried to be as descriptive as I can, please let me know if you need any other information.

Thank you,

Read More

Abcam community

Verified customer

Asked on Aug 24 2012

Answer

Thank you for contacting us.

This kit has been strictly quality checked andwe haven't received any quality complaint so I am sorry we are not sure where the problem might be.

Have you stored the standard at -20C as recoemnded or at 4C?
Have you checked the background or blank readings?

I will look forward to hearing from you soon.

Read More

Abcam Scientific Support

Answered on Aug 24 2012

11-20 of 37 Abreviews or Q&A

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