Here is whole protocol for measuring the SOD with concentration of each chemical. But quantity is not mentioned.
Superoxide dismutase activity was estimated spectrophotometrically by following the method given by Misra HP and Fridovich, 1972.
The method is as follows.
The reaction mixture of 3 ml was prepared by adding
50mm potassium buffer,
45 μM methionine,
5.3 μM riboflavin,
84 μM NBT, 20 mm potassium cyanide and
500 μl of tissue homogenate.
The mixture was maintained at 25 ºC equipped with 15W fluorescent lamps in an aluminum foil-lined box.
After the incubation period for 10 min, reduced NBT was then measured using Spectrophotometer at 600 nm.
One unit of enzyme activity was defined as the amount of enzyme giving a 50% inhibition of NBT reduction.
The enzyme unit can be calculated using the following equations: Rate (R) = Final OD-Initial OD / 3 min % of inhibition = (Blank OD-R / Blank OD) × 100 Enzyme unit (U) = (% of Inhibition / 50) × common dilution factor. (50 % Inhibition= 1 U)
In above method I want to know the quantities of each chemical. I Understood the concentrations but how to find defined quantity, is still a big issue for me.
Anyone can please tell me the quantity and method to derive quantity if its not given in article.
It would be better to follow recent, modified methods, especially, kit-based methods as they are straight-forward! You could also infer your information from them. Check these out including attachments:
https://www.sigmaaldrich.com/SA/en/product/sigma/cs0009?gclid=EAIaIQobChMIueDOs7ml8QIVkeR3Ch2VEAciEAAYASAAEgJJPPD_BwE#
A simple method for clinical assay of superoxide dismutase.
Y Sun, L W Oberley, Y Li. Clinical Chemistry, Volume 34, Issue 3, 1 March 1988, Pages 497–500, https://doi.org/10.1093/clinchem/34.3.497.
Thank you Riaz A. Khan sir for your answer. But the issue is, we have all the chemicals listed in the method and we can not afford the kit. The cost is much more higher. Still I am grateful for your time.
I suggest you look closely into the ratios of the materials used based on their stoichiometry in these methods and infer information for your reagent materials and the analyte sample according to the current stoichiometry.
Maybe change your method to Giannopolitis and Ries (1977)
PLease see my paper in MAYDICA journal
The reaction mixture contained 100 μl of 1 μM riboflavin, 100 μl 12 mM L-methionine, 100 μl 0.1 mM EDTA (pH 7.8), 100 μl 50 mM Na2CO3 (pH 10.2), 100 μl 75 μM nitroblue tetrazolium in 25 mM sodium phosphate buffer (pH 6.8) and 200 μl crude enzyme extract, in a final volume of 3 ml. Glass test tubes that contained the reaction mixture were illuminated with a fluorescent lamp (120 W), and identical tubes that were not illuminated served as blanks. After illumina-tion for 15 min, absorbance was measured at 560 nm. One unit of superoxide dismutase activity was defined as the amount of enzyme which caused 50 % inhibition of photochemical reduction of nitroblue tetrazolium.
Dear Vijay Kevlani ,
The summary of chemicals can be found in a number of sources. The most reliable is: Article A comprehensive review on the determination of enzymatic ass...
in “section 4.1 | Superoxide dismutase assay you can read” and I quote “The final volume of incubation mixture is 3 ml that contains 50 mM of potassium phosphate buffer (pH 7.8), 45 μM of methionine, 20 μM of potassium cyanide, 84 μM of nitroblue tetrazolium (NBT), and 5.3 mM of riboflavin”.
This way you can mix the chemicals together such that the end concentrations are according to this protocol.
Best regards.
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