I tried to measure the activity of superoxide dismutase (SOD) in bone homogenate (10%)....
I found a good paper that took Marklund method as reference with some modification
Basic & Clinical Pharmacology & Toxicology, 103, 55-60 (2008)
Sandra Maniam, Norazlina Mohamed, Ahmad Nazrun Shuid and Ima Nirwana Soelaiman
Palm Tocotrienol Exerted Better Antioxidant Activities in Bone than
the paper said: 2 ml 0.01MTris-HCl buffer pH 8.5 + 750 ul sample + 750 ul water + 500 ul pyrogallol (2mmol in 0.01M HCl)
and read the absorbance at 420 nm at 1,2,3 min then calculate dA/dt, % inhibition, SOD concentration then its specific activity.
My problem is the difference between readings at intervals (1,2,3 min) for the sample were larger than those for the blank
as I calculate the % inhibition from the equation:
% inhibition= 100 - (dA/dt for sample / dA/dt for blank * 100)
so the % inhibition gave a negative result (unreasonable one)
i changed alot of factors including the sample volume and pyrogallol concentration.... but all trials were in vain...
please, is there anyone can help to solve this problem....