Western Blot: Mitochondrial Extraction Kit [NBP2-29448] - Mouse liver cells were fractionated using . 20 ug of cytosolic (lane 1) and mitochondrial (lane 2) extracts were probed with anti-Cytochrome C antibody, and ...read more
The procedure described here for the isolation of mitochondria is based on the principle of differential centrifugation. The high density nuclei are first removed by low-speed centrifugation on a sucrose cushion. The supernatant containing the mitochondria is subjected to high speed centrifugation to retrieve the mitochondria. This kit contains reagents required for the isolation of mitochondria from 10 grams of mammalian tissues (liver, muscle, etc.) or 100 reactions for cells grown in tissue culture plates. The procedure describes isolation of mitochondria from 1 gram of starting tissue material (mouse liver) or 5 x 107 HeLa cells. The Mitochondrial and Cytosolic fractions can be used for studying apoptotic and signal transduction pathways to detect translocation of any factors of interest within the two fractions by western blotting, ELISA, or other assay.
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FAQs for Mitochondrial Kit (NBP2-29448). (Showing 1 - 1 of 1 FAQs).
We are looking to isolate mtDNA that is free from nuclear DNA and nuclear DNA that is free from mtDNA. Do you know if these kits (NBP2-29447, NBP2-29448) will be able to do this? We are wanting the isolated DNA to use as a positive control for a copy number experiment.
You would need to use both kits in order to seperate mtDNA and nDNA. I was worried about the lysis buffer contained in both kits would force the DNA to precipitate, this can occur if the buffer is very basic or alcohol based. I also double checked to make sure all the buffers contained within the kit do not have any DNAses or RNAses present. Follow the protocol for both kits until you use the mitochondrial/nuclear lysis buffer and then proceed to collect the individual supernatants. I would proceed to use our Quik ChIP Kit. The way this kit works is by precipitating the DNA and then running it through a spin column included into the kit. Then the DNA is eluded of the column and the resulting product should be purified mDNA or nDNA depending on which supernatant you used. I reccomend on using a lot of starting material for your nuclear extract as the yield tends to be low, which is typical. I also reccomend to use 18 mega ohm DI water when starting your nuclear/mitochondrial extractions. The nuclear and mitochondrial extraction kits were designed for the purpose of extracting proteins and have not been tested for this particular application.