Hi everyone

i did this protocol to measure the anti-lipid peroxidation activity of plant extracts.

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. Briefly 100 μL of bovine brain extract sigma (B1502) was mixed with a reaction mixture containing 30 μL of buffer, (100 μL) of (1- 1000 ug/ml) extract or positive control vitamin C , and 30 μL 5 mM sodium nitroprusside (SNP) as the prooxidant. The volume was made up to 300 μL by water before incubation at 37°C for 2 hrs. The colour reaction was developed by adding 300 μL 8.1% SDS (sodium dodecyl sulphate) to the reaction mixture, this was subsequently followed by the addition of 500 μL of acetic acid (pH 3.4) mixture and 500 μL of 0.8% thiobarbituric acid (TBA). This mixture was incubated at 100°C for 1 hr. Thiobarbituric acid reactive species (TBARS) produced were measured at 532 nm.

source:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564280/

The problem is that the generated pink colour of chromogen increases with increase the positive control concentrations (vitamin C). where the control the brain extract + sodium nitroprusside (SNP) as the prooxidant gives the lightest pink colour.

Any suggestion?

Thanks in advance...

Maryam