Total Antioxidant Capacity Assay Kit (ab65329)

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.

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.

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.

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.

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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.

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.

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.

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.
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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.

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.