I stored small pieces of rat colon in PBS at -20 and now I want to do SOD and MDA assay, I also need to do the Bradford assay to quantify the protein content in the samples. can anyone tell me which steps to follow? or a protocol you think is going to be helpful?
Dear Ifeoma!
Please You look at this article and protocols:
Article Measurement of superoxide dismutase, catalase and glutathion...
Article Increased lipid peroxidation, apoptosis and selective cytoto...
(attached)Cell culture The human colorectal adenocarcinoma cell line LoVo and its doxorubicin-resistant subline LoVo/Dx
2.5. Lipid peroxidation assay
Cells were seeded (800 000/well) onto a six-well plate in 2 ml of medium and incubated for 24 h at 37 °C. Then, the studied compounds (5–100 μM) were added in a fresh portion of medium, and the incubation was continued for 48 h. Cells were scraped, centrifuged (13 000 x g, 10 min, 4 °C) and washed with phosphate-buffered saline (PBS). The amount of lipid peroxidation product malondialdehyde (MDA) in the samples was measured using lipid peroxidation assay kit (Abcam) according to the manufacturer’s protocol. Shortly, the lysed
cells were reacted with thiobarbituric acid (TBA) to generate the MDATBA adduct. The MDA-TBA adduct was detected colorimetrically (A532). The amount of MDA equivalents [nmol] in samples was estimated by interpolation from the standard curve. The control sample was treated with no PHDs.
Cellular protein concentration in the supernatant was determined by Bradford reaction.
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