Cell fractionation: nuclei do not pellet - (Jun/07/2007 )
I am trying to fractionate cytoplasm and nuclei by douncer homogenization followed by nuclei purification. The last step in my protocol consists of layering nuclei suspended in 0.25M Sucrose on top of a 0.35M sucrose cushion in a standard 15ml tube and spinning at 1430g for 5min in a swinging bucket centifuge. I understand this is a common way to isolate nuclei. However, during this last step the nuclei fail to pellet but gather on the side of the tube and when looking under the microscope the nuclei still seem to have chunks of cytoplasm attached to them.
I have tried changing tubes, centrifuge and varying the acceleration of the centrifuge, but with no change in the outcome.
If anyone has had similar problems or have possible solutions, I would very much appreciate hearing them.
University of Copenhagen
How do you get the nuclei to stick to the sides? If you are using a swinging bucket rotor, make sure that the arms swing out correctly. Most likely this was the first thing you checked but this is the only thing I can think of. It certainly is a mystery...
Yes, the arms swing nicely but just in case I also tried another centrifuge with the same result. I am wondering, whether the nuclei are deposited on the side, when the buckets swing out. However, it should be a common method for fractionation, other people in the lab had the same problem, but I haven't heard of anyone else with this problem, so it seems we are doing something consistently wrong.