mitochondria isolation - sucrose gradient (help, please!) - (Apr/15/2006 )
I'm having problems purifying mitochondria by a discontinuous 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 250 mM sucrose to pellet the mitochondria, which are supposed to be in the interface fraction. Unhappily, we find no mitochondria in any fraction, when before the ultracentrifugation step we have a heavy membrane pellet!!! I'm thinking of using bigger ultracentrifuge tubes, since most protocols use higher volumes (for example 18 ml of each layer or the gradient), but I will thank any help, before buying them.
I'm desperately waiting any comment. Thanks in advance.
have you checked the pellet for mitochondria? you may be too concentrated and the heavy membrane fraction may be carrying the mitochondria into the pellet.
try using a lower volume or lower concentration of sample in each tube.
Thanks for your reply. Unhappily I'm not sure to understand you or I'm afraid i didn't explain my problem very well.
The heavy membrane pellet is what I load in the sucrose gradient (after resuspension, obviously). This heavy-membrane pellet is obtained after centrifuging at 10000g the post-nuclear supernatant and it's supposed to be highly enriched in mitochondria (in fact, it is used as a mitochondrial fraction in many papers).
I have checked by western blot that the heavy-membrane pellet is highly enriched in mitochondrial markers, but It also contains lysosomal proteins (this is the reason for trying a sucrose gradient).
Anyway, after the ultracentrifugation of the sucrose gradient there is no pellet. I have collected all the fractions in the sucrose gradient and after diluting to 250 uM sucrose and re-centrifuging at 10000g to pellet the mitochondria I see no pellet again.
i'm going to preface this response with: please don't be insulted by any of the following.
i assume you are using a swing bucket rotor for your gradient (if not, check the walls of the tube for your markers). when you set your buckets on the rotor, how far do you tip the buckets to ensure that they are properly set? if you tilt too far, your sample will pour out and you will find little to none of your markers in the fractions, but you will find them in the bucket. check your buckets for your material. this happened to a friend of mine and it was not resolved until i saw his manipulation and pointed it out (he didn't believe me until after the spin i showed him all of his sample in the bucket).
instead of tipping the buckets you can waggle them from side to side to ensure that they are set properly.