What kits are you trying to use?

Through ELIZA or Nanodrop?

Dear Hlahasoane

Can u plz share the manual.

Download this article (Effect of crocin on antioxidant gene expression, fibrinolytic parameters, redox status and blood biochemistry in nicotinamide-streptozotocin-induced diabetic rats) and you will find the answers of your questions.

Please, read this papper:

doi: 10.1038/nprot.2009.197. Epub 2009 Dec 17.. 2010 Jan;5(1):51-66.Nat Protoc

Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue

Christine J Weydert 1, Joseph J Cullen

PMID: 20057381. PMCID: PMC2830880. DOI: 10.1038/nprot.2009.197

• Good luck

Check my recent publications on assay of antioxidant activity and probably share the manual of the kits you are using.

For GSH: You should use L-glutathione as a standard. For prepare standards, you should prepare 1 mg/ml GSH, Elmann reactive in Tris pH 7, Tris HCl pH 8.4. The final volume of the bathtub should be 2.2 ml: 2 ml Tris 8.4, 0.1 ml Ellmann, 0.1 ml GSH dilution. (For blanks, you should use distiled water instead of GSH) And then, you should make up dilution and measure 412 nm. You should use TCA (%5) as a buffer in homogenization for the sample (for 1 g sample, 4 ml TCA solution). After, you should set the pH of your sample to 4-6.

The final volume of the bathtub should be 2.2 ml again, but this time you should put your sample instead of GSH.

Good luck!

https://scholar.google.com/citations?view_op=view_citation&hl=en&user=br2OROUAAAAJ&citation_for_view=br2OROUAAAAJ:tOudhMTPpwUC

Read this article.

Enzymatic antioxidants

These include enzymes such as superoxide dismutase (SOD).

Glutathion peroxidase (GSH-px), peroxidase, catalase (CAT), catalase, and reductase glutathion (rd-GSH), and these enzymes have a retrograde effect on their natural level in a way that removes the toxicity of free radicals, directly or indirectly through their control over levels of Some ions such as iron and copper.

1-(SOD) Super Oxide Dismutase Enzyme.

The function of this enzyme is to restore the vitality of cells and reduce the speed of their destruction.

Free radicals, and this enzyme also helps in its science to benefit from copper, zinc and manganese.

The levels of this enzyme tend to decrease gradually with age (at the time when it increases).

Its potential as an anti-aging treatment is currently under research. There are two types of free radical production

This enzyme is a type with copper and a type with zinc, and each of the two types works to protect a special part of the cell.

The first works to protect the cell cytoplasm, where free radicals are produced as a result of the activities

different metabolites, and the other one is effective in protecting the mitochondria of cells that contain

The genetic information of cells and serves as a site for energy production, and the enzyme SOD is one of the

Enzymes involved in the loading of toxic products of cellular metabolism, and it removes oxygen radicals

O2 by accelerating the rate of conversion to H2O2 with the help of some minerals such as semenium and copper.

2- Catalase Enzyme.

CAT enzyme is found in most organisms and in all organs of the body and is concentrated in the liver

red blood cells, kidneys, and in small quantities in the brain, heart, and muscles, and works to get rid of

H2O2 by converting it into H2O and O2, so it prevents the accumulation of hydrogen peroxide produced

One of the processes of respiration in tissues, which causes the accumulation of cell poisoning and then death.

Catmase is present in all organisms with aerobic respiration and in cells that contain cytochrome c.

It is one of the first enzymes to be studied and purified, as bacteria, birds and livers of birds were studied.

Animals, as well as human liver, were purified in large quantities, due to their vital activity.

The enzyme catalase is one of the important enzymes in the tolerance of H2O2 generated during gestation and is

It represents the second stage of the defense system in plant cells and tissues after the SOD enzyme

H2O2 is taken up and converted into a molecule of water and oxygen, and the Cat enzyme is present in certain organs such as

Peroxisomes and Glyoxysomes as well as in mitochondria and in the cytoplasm and plastids.

This enzyme is characterized by its ability to use hydrogen peroxide as a acceptor and a donor at the same time.

That is, the action of this enzyme is to oxidize a molecule of hydrogen peroxide with another molecule of it.

Two molecules of hydrogen peroxide participate in the interaction with the catalase enzyme.

as a hydrogen donor and the other molecule acts as a LO acceptor, i.e. one molecule is oxidized and one molecule is reduced

Another form of hydrogen peroxide to give oxygen and water. The catmase enzyme is composed of

Proteins that contain an iron group and have three isoenzymes, Cat1, Cat2, and Cat3.

It has become clear through extensive studies that the effects of the types of good will t is clear

The catmase enzyme is clearly affected in reducing damage to antioxidant systems, specifically

In different hardships, such as finding water, as well as salty hardening and reconstruction cases. And to increase the roots

Free radicals have the effect of decreasing the activity of CAT and other antigenic enzymes and thus cause an increase

In hemolysis and cytotoxic effects by oxidation of membrane lipids and cytokine proteins.

3- Glutathione Peroxidase (GPX) Enzyme.

The enzyme kumtathione peroxidase is prevalent in many apocrine species, where it is localized in

Mitochondria and cytosol are two of the most important antioxidant enzyme systems due to their ability to

Removal of many free radicals and hydroperoxides resulting from oxidation.

Clotathione peroxidase is an enzyme with a molecular weight of 44,000 D and is a seminoprotein.

Selenoprotein is present in the cytoplasm and mitochondria of liver tissue and red blood cells.

And plasma and sperm.

Great in regulating the level of various peroxides by accelerating

The enzyme (GPX) leads to a

GSH (glutathione Reduced) is converted to oxidized glutathione.

GSSG (glutathione Oxidized) after removing various peroxides (such as peroxide

hydrogen or fat peroxides (LOOH) or organic peroxides (ROOH) as in

(equations in below), where GPX represents the antioxidant and by-products of the cell protection system.

part

Reduces cell damage caused by excess free radicals (oxidative stress).

2GSH + H2O2 GPX GSSG + 2H2O

2GSH + LOOH GPX GSSG + LOH + H2O

2GSH + ROOH GPX GSSG + ROH + H2O

The presence of semenium in the composition of the enzyme kemutathion peroxidase works to protect the components of the cell.

and biological membranes from oxidative damage. Therefore, exposing the body to oxidative damage leads to

The significant decrease in the level of kmutathione peroxidase, which indicated a significant decrease in the level of the enzyme kmutathione peroxidase in the blood serum of mice exposed to oxidative stress induced by methotrexate through subperitoneal injection. Cofactors are important for enzyme activity, so a decrease in the concentration of copper in the blood can reduce the activity of the enzyme, in addition to the fact that in the case of hypothyroidism, the activity of the enzyme decreases.