You're going to need to provide more information about your sample prep and assay.

@John Carter Thanks for your valuable response. According to the protocol mentioned below, I had performed the LPO assay, but there is no subsequent colour change occurs at the end.

Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351-8.

Reagents Blank (ml) Test (ml)

Thiobarbituric acid 1.5 1.5

SDS (8.1%) 0.2 0.2

20% Acetic acid 1.5 1.5

Sample * 0.2

Distilled Water 0.8 0.6

Kept in boiling water bath at 90 C for 1 hour and cool in tap water

Distilled water 1.0 1.0

Read @ 532nm

This procedure was followed by me based on Ohkawa et al., 1979, except n-butanol:pyridine composition. Is this the procedure is correct? If it not please mentioned some suggestion, sir.

Thanks in advance.

The easy way to make this reaction completely fail is pH is >3. Check the pH of your assay negative control mixture before and after heating.

Otherwise high concentrations of amino acids, proteins, or nucleic acids in the sample can sequester the aldehyde assay targets.

The organic solvents are used to extract the dye complex away from substrate that may cloud the solution interfering with absorbance reading. If your samples are not cloudy the organic step should not be necessary. It's not important for color development.

Do you have a positive control? Most people use 1,1,3,3-tetramethoxypropane, which is a protected but reactive form of malondialdehyde.

John Carter Thank you again, sir. I'll rectify it sir. I will look forward the above mentioned notes.

Another doubt is, what percentage of tissue sample is required 1% or 10% for LPO assay?

And Is the LPO protocol mentioned above previously are good one?

Thanks in advance.