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oxidative stress protocol


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#1 jasmina

jasmina

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Posted 07 October 2010 - 12:50 AM

I would like to measure the level of oxidative stress in mouse brain tissue. i read that there is a way to study some antioxidants enzymes such as Superoxide dismutase, catalase, glutathion synthetase..
Do you have any ideas about the technique to use and any potential simple protocol to do this??
thanks for help

#2 Zagami Francesco

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Posted 15 June 2017 - 12:38 AM

Fluids estimation of superoxide dismutase using pyrogallol method modified by myself

Principle

Pyrogallol autooxidises rapidly in aqueous or alkaline medium solution and this has been employed for the estimation of superoxide dismutase. SOD inhibits the auto-oxidation of pyrogallol. This principle was employed in a rapid and convenient method for the determination of the enzyme concentration.

Reagents

Pyrogallol solution : dissolve 303 mg (24 mM) of pyrogallol in 100 ml of 10 mM HCl (dilute 83.2 µl of concentrated HCl conc. to 100 ml with dH2O) obtain a acid pH medium in which autoxidation of pyrogallol is inhibited.

Assay buffer : in 70 ml dH2O, dissolve 605 mg (50 mM) Tris nd 0.0372 g (1 mM) EDTA-Na22H2O, adjust pH to 8.2 with about 50 µl of concentrated HCl and fill up to 100 ml with dH2O. Before the use, shake vigorously the assay buffer to give appropriate aeration to it

Catalase Assay buffer : before to use mix 33 µl of (10 µM) bovine catalase solution (2.5 mg protein/ml) per ml of assay buffer

Protocol for tube

Dispense 20 µl of sample to tube, then add 950 µl of catalase assay buffer, mix and then add 30 µl of pyrogallol solution. In EIA reader tube, measure Abs with wave length 420 nm, at 25 °C, in kinetic program conditions, with Abs readings every 10 sec by 3 minutes.

Protocol to microplates

Dispense 6 µl of sample to assay cell, then add 289 µl of catalase assay buffer, mix and then add 9 µl of pyrogallol solution. In microplate reader, measure Abs with wave length 420 nm, at 25 °C, in kinetic program conditions, with Abs readings every 10 sec by 3 minutes.

Formula for activity calculation 

 

 

                                                  (Abs/min(blank) – Abs/min(sample)) *sample dilution *assay volume

                      Units/ml =  ------------------------------------------------------------------------------------------------    

                                            sample volume *molar extinction coefficient 0.0436 mM (for path length 1 cm)

 

 

I hope that this will helpful for You. If you want for other enzymes, contact to me: francesco_zagami@virgilio.it






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