I am looking at protocols to do cell fractionation in TISSUE. I want to detect the expression of different proteins by Western Blots on both cytosolic and nuclear fractions. Can anyone point me to a protocol which works well?
by differential centrifugation you can separate nuclear and cystolic fractions.
Hi Modar,
are you planning to use commercial kit or home made lysis buffer. Kits are very expensive and teh home made one works just as well. Even if you use a kit, you have to optimise the conditions. How you process your tissue depends on the type of tissue you are planning to use as some tissue are easier to dissociate than others.
Then your buffer can either use high salt to pop the cells or detergent or both. However, with tissue you also need to mechanically disrupt the tissue. A dounce homogeniser from sigma helps you separate the different organelles without popping them (mitochondria, nucleus). The number of times you sheer the tissue with the homogeniser needs to be optimised. Ie. after each 5 sheers put a very small drop of the cell mixture onto a glass slide and visualise it under a light microscope. You would see large round organelles (nucleus) and smaller round organelles (mitochondria) floating around. You would also need to optimise how much buffer to add to the tissue as too much reduces ypour recovery and too little means contaminated fractions. Then you do differential centrifugation. This step also needs to be optimised though start with speed recommended in publications. About 350g for 10min at 4 degrees removes unbroken cells and debri. Then centrifuging the supernatant for about 10min at 850g sediments out nuclei. The supernatant then spun at 12,000g for 30 min separates out mitochondria.
Look at:
Fumitaka et al Transplantation. 72(11):1803-1807, December 15, 2001
Bile Salt Tauroursodeoxycholic Acid Modulation of Bax Translocation to Mitochondria Protects the Liver From Warm Ischemia-Reperfusion Injury in the Rat1
Cheers
thank you all for the answers, I guess i will start with this protocol
Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.
Cox B, Emili A.
Nat Protoc. 2006;1(4):1872-8.
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