Hi,

I have previously optimized an assay for SOD using xanthene/ xanthene oxidase (XOD) system in order to characterize two recombinantly expressed SODs from a mollusc and a fish species.

You can refer to the following articles:

https://www.researchgate.net/publication/231611768_A_manganese_superoxide_dismutase_with_potent_antioxidant_activity_identified_from_Oplegnathus_fasciatus_Genomic_structure_and_transcriptional_characterization?ev=prf_pub

https://www.researchgate.net/publication/229080782_A_manganese_superoxide_dismutase_(MnSOD)_from_Ruditapes_philippinarum_Comparative_structural-_and_expressional-analysis_with_copperzinc_superoxide_dismutase_(CuZnSOD)_and_biochemical_analysis_of_its_antioxidant_activities?ev=prf_pub

The assay is based on the competitive use of superoxide ions (which was generated by XOD during the conversion of xanthene into hydrogen peroxide and uric acid) by SOD in your interested tissue samples and NBT which subsequently forms a dye (NBT-diformazan).

You have to be prepared with the following:

1. Nitro Blue Tetrazolium (NBT): Sigma offers tablets and you may prepare a 0.75 mM solution

2. Xanthene: Sigma offers, you may prepare a 30 mM stock and could dilute 10 times just before performing the assay

3. EDTA: You could prepare 0.1 M solution and dilute into 3mM solution just before performing the assay

4. Bovine Serum Albumin BSA: Sigma offers; and you suppose to have a 0.15% (W/V) working solution

5. 6 mM CuCl solution (To terminate the reaction) [What I did was to incubate the assay for 20 min, so that reaction could be considered to be completed].

6. Xanthene oxidase: Sigma offers (As per the manufacturer’s instructions, you have to do a simple calculation and dilute the XOD and you have to add 6 mU for each assay (Better to keep in ice).

7. Finally have your tissues to be tested.

If you want to optimize the reaction pH, you can perform a separate optimization assay (refer the articles); if not you could choose the physiological pH (7.4-7.8).

Other than these stuffs, you may need to have a multi-plate reader and other common stuffs to conduct the assay.

Assay could be performed, as per the volumes indicated in the articles I mentioned.

Good luck.

Article A manganese superoxide dismutase with potent antioxidant act...

Article A manganese superoxide dismutase (MnSOD) from Ruditapes phil...

Thanks a lot!

This assay is to quantify the activity of SOD right?

If I want just to inhibit O2- production by SOD  can I prepare a stock solution and then add to the final  the reaction mixture? I have to make some experiments with my cells and I need as a control to inhibit the O2- production through SOD. Can I add directly  SOD to my reaction?

 for SOD follow the method of

 Beauchamo, C.O. and Fridovich, I. (1971). Superoxide Dismutase: Improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287.

I didn't explain well what's my plan. I want to buy the SOD powder fromSigma and make a solution and add to my cell mixture to inhibit the O2- production. I would like to know if someone knows the right concentration of SOD to be used to inhibit the O2- production. Which solvent is used to dissolve SOD powder?

Can someone give me a procedure for Superoxide Dismutase estimation in serum. Since it doesn't need extraction of hemosylate from RBC's can u you give serum specific procedure?

@ Neelam Pawar

You can use DetectX® Superoxide Dismutase Colorimetric Activity Kit

(Catalog Number K028-H1) to estimate the serum SOD. Simply click the following link,

http://www.funakoshi.co.jp/data/datasheet/ARB/K028-H1.pdf

Alexandra I understood your question and I have the same problem please tell me if you got any methods for the best buffer solution can we use to homogenize the liver to estimate antioxidant enzymes ? because I have small liver sample and the same kits assay of  Cayman'.

Dear Neelam,

You can measure serum SOD activity as described in the attached document, I used this method to measure the activity in hundreds of human plasma/ serum samples.

To,

Mostafakamal Shams ·

I would suggest you to go through a review article on SODs by Dr. Fukai.

ANTIOXIDANTS & REDOX SIGNALING

Volume 15, Number 6, 2011

Superoxide Dismutases:

Role in Redox Signaling, Vascular Function, and Diseases

Karima Zitouni

Will you please mail me your lab protocol for SOD measurement in human plasma /RBC that would be of great help

[email protected]

Can anyone provide me the protocol of quantitative estimation of SOD in plant tissue?

You can mail the protocol in my personal mail id([email protected]).Thanks in advance.

Hello! i have a question. i want to know how must i calculate the SOD activity with an essay with Pirogalol

Rahul Shivaji Patil

please sent me protocol of sod colorimetric determination