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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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mitochondria purification by sucrose gradient (help, please!)
Posted by: AUG (IP Hidden, New member, 1)
Date: April 15, 2006 11:41AM
Hi, everybody!
I'm having problems purifying mitochondria by a discontinouos sucrose gradient, so I would thank any help. We usually load the heavy membrane fraction obtained by differential centrifugation onto a 1-1,5M discontinuos sucrose gradient (1,75 ml of sucrose 1,5 M, 1,75 ml of sucrose 1M and 500 ul of the heavy membrane fraction in a buffer containing 250 mM sucrose). Then we ultracentrifuge at 60000g for 20 min (4ºC). Finally we collect several fractions of 0,5 ml and dilute them to a final 250 mM sucrose concentration to pellet the mitochondria (10 min, 10000g). Mitochondria are supposed to be in the interface beetween the two layers, but we find no mitochondria in any fraction, when before the ultracentrifugation step we have a heavy membrane pellet!!! I'm desperately waiting any comment. Thanks in advance. Edited 1 times. Last edit at 04/15/06 11:45AM by AUG.
Re: mitochondria purification by sucrose gradient (help, please!)
Posted by: VDEP (IP Hidden, New member, 3)
Date: October 26, 2007 11:31PM
I just saw your posting; had the same problem with my first purification. Did you ever find a solution to the "missing mitochondrial pellet" question?.
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