I'm trying to determine oxidative stress levels in plasma of fish. The problem is I only have about 500µl of plasma for each individual and the lipid hydroperoxide levels is just one of the parameters. Will 150µl be enough?
I'm afraid using this small amount of sample will go beyond the detection sensitivity of the kit.
Try to dilute your samples and use the dilation factor in your calculation to get the exact value of your real samples
We have used this procedure and the key is the extraction process with chloroform. You may not have enough of the volume you need since you have a small volume to start. I would suggest trying an extraction with a known concentration of lipid hydroperoxides first in the volume you anticipate to extract and check out your recovery rate. See if you can then dilute that concentration into a larger volume and see if you obtain a similar amount and recovery. This will let you know if you can dilute the sample and extract the lipid hydroperoxides. We have used this kit and the rest after the the extraction is not difficult. Need to use glass pipettes to get the extracted sample.
We tried to use the kit in mouse plasma samples a couple of years ago, and the results were unreliable. maybe doing what Allan suggests, would help since the chloroform extraction is critical.
Do not waist money on this type of kits. Think a little and you will do the method yourself.
The lipid is already soluble in plasma so there is no need to extract. A key issue is background absorbance. Be sure to have a well or two as sample blanks (sample without reagent).
Thanks for your answers.
Alexander I'm actually looking for ways to replace this kind of kit. Do you have any suggestion for the LPO kit?
I have used small volume of sample for my experiment and everything was fine. if you decrease ur samples volume you have to decrease the reagent volume as well. thats what I did. for example if you are using 100 ul that means you have to decrease your reagent 5 folds as well
Even i am also agree with pouya suggestion , because if you want to reduce volume of sample then ther is no problem to reduce volume of reagent , which is not going to effect the sensitivity of the reaction or alternative mehod dilute tha samle 1;5 os 1;10 ratio and can be used and in final calculation muliply with that diluted multification factor , and another thing is that for LPO estimation always should be used fresh sample, better not to use storage sample for more appropriate result
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