I want to measure lipid peroxdation level in mouse colon tissue homogenate. Can anyone help me out in this?
You can find a relevant protocol in the 3rd page of the below pdf file.
Hi! Good protocol
Biochemical assays
Thiobarbituric acid reactive substances (TBARS) assay.
TBARS were measured by the method of Kamyshnikov (2004). Briefly, to 2 mL of distilled water was added to 0.1 mL of tissue, 1 mL of 20% trichloracetic acid (TCA) and 1 mL of 0.8% 2-tiobarbituric acid (TBA) reagent, and boiled in a water bath at 95 oC for 10 min. The reaction was stopped by placing the tubes in ice-cold water. Then mixture was centrifuged at 3,000g for 10 min. The absorbance of supernatant was read at 540 nm. The µmol of TBARS was calculated by using 1.56∙105 mM-1∙cm-1 as extinction coefficient. TBARS level was expressed in µmol of MDA per mg protein.
Kamyshnikov VS (2004) Reference book on clinic and biochemical researches and laboratory diagnostics. MEDpress-inform, Moscow
You can discuss method of Ohkawa et al (1979) for lipid peroxidation (LPO) which have been a paradigm method in the field. There are many modified versions now, for example, one that measure LPO in cultured cells and other that measure in tissue homogenates. You can run MDA standard (1,1,3,3-tetramethoxypropane) also while using molar extinction co-efficient (of MDA-TBA complex) that will help you in standardizing this measurement. Since you are using tissue homogenate, you will have to ensure that tissue matrix is fully digested ( by blending and use of digestive enzymes like trypsin or chymotrypsin in tissues) before TBA addition in the supernatant obtained.
Hİ
You can use our protocol according to this article: Abdulahad Doğan & İsmail Çelik (2016) Healing effects of sumac (Rhus coriaria) in streptozotocin-induced diabetic rats, Pharmaceutical Biology, 54:10, 2092-2102,
Are you making homogenates with lipid peroxidation inibitors like BHT and iron chelators such as DEPA? If not, your homogenization is likely causing artifactual lipid peroxidation. TBARS is a poor way of measuring lipid peroxidation is tissue because of artifacts and has many technical caveats. You can measure HNE-protein adducts by western, but then again you need to guard against lipid peroxidation during tissue processing.
Yes
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I think this article will help you to sort out your problem. To estimate lipid peroxidation in tissue homogenates, use tissue homogenate insteed of using blood. Rest you need to follow the same protocol. Feel free to ask questions regarding lipid peroxidation.
thank you
Please see the attached PDF.In our hands this method based on TBA works best.
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