Q: Can I use your TBARS Assay to measure the TBARS level in my blood?
A: No, our TBARS Assay Kit is strictly for use by researchers in a laboratory. It cannot be used for diagnostic use to test patient samples.
Q: How does the sensitivity of your MDA ELISA Kits compare to the TBARS Assay?
A: The MDA Adduct Competitive ELISA Kit is more sensitive than the TBARS Assay, but actually the two assays are not directly comparable. The MDA ELISA is more specific and only measures MDA protein adducts, while TBARS measures total MDA, including free MDA and MDA adducts, resulting in higher values with the TBARS assay compared to the MDA Adduct ELISA. If you are using samples that have a high hemoglobin content, you may want to consider using the ELISA since hemoglobin can interfere with the TBARS Assay.
Q: What is the butanol extraction?
A: Butanol extraction is an optional step performed on both the samples and standards. It is used to remove any remaining hemoglobin in the sample that could cause a false positive due to hemoglobin’s strong absorbance at 540 nm which is very close to MDA-TBA absorbance at 532 nm. The pink color of the solution after the MDA-TBA adduct reaction can come from either the MDA-TBA or hemoglobin, so performing a butanol extraction allows you to measure just the color initiated from MDA-TBA adduct.
Q: Is the butanol step necessary?
A: The butanol extraction is an optional step that should be performed on serum samples or other samples that have high hemoglobin levels. The butanol extraction step will remove hemoglobin from the sample, which has the same absorbance as the MDA-TBA complex and can cause a false positive if present in the sample.
Q: Is the butanol fraction the upper phase?
A: The butanol will be the upper (organic) phase after vortexing and will contain the MDA-TBA. Hemoglobin, which is protein, will stay with lower (aqueous) phase.
Q: Why does your kit manual recommend using samples less than 1 month old?
A: The storage guidelines refer to the MDA modification, which is not very stable, and can begin to degrade after 1 month of storage at -80ºC. Degradation may not be an issue if you are only interested in relative comparisons between samples that have been stored for the same length of time. It may be more difficult to detect MDA in older samples, but this will depend on the initial level of MDA and the extent of degradation.
Q: What can I do if my samples have been stored at -80ºC for over a year?
A: For samples that have been at -80ºC for a year, you may want to consider testing a different marker such as 8-OHdG in DNA or protein carbonyl, both of which are very stable markers of oxidative stress.
Q: Is it necessary to add BHT to samples?
A: BHT is an antioxidant that helps prevent samples from further oxidation during the assay and we don’t recommend omitting this from your samples.
Q: Is this compatible with liver tissue samples?
A: Liver samples can be used with the TBARS assay; however liver contains many red blood cells which can interfere with the assay. When using liver samples with the TBARS assay, we recommend performing both the perfusion and butanol extraction steps to remove any remaining hemoglobin that can produce a false positive due to hemoglobin’s strong absorbance at 540 nm.
Q: Is this assay compatible with plant samples that contain anthocyanins?
A: We have not tested the TBARS Assay with plant samples, but it should be compatible. The butanol extraction (step 8 of the assay protocol) should be performed, which will remove the anthocyanins to the aqueous phase. There is evidence in the literature that the TBARS Assay has worked with plant samples.
Q: What is the best way to collect serum and plasma samples?
A: There is no specific collection protocol for use with the TBARS assay and any anticoagulant can be used. Samples should be used soon after collection since MDA is not stable, and samples can only be stored at -80 C for up to 1-2 months.
Q: Is the TBARS Assay compatible with samples of any species?
A: Our TBARS Assay is not species specific and can be used with samples from any species.