I've been trying to isolate extracellular vesicles on an optiprep gradient in 5-ml tubes. I've tried 2 and 12 h centrifugation and in both cases, the fractions seemed to be mixed after centrifugation and the vesicles mostly remained on top of the gradient. I never have this problem when I use 38-ml tubes (SW32 Ti rotor) for 2 h but for a particular experiment I need to run this in small volume. What factors should I change in my protocol when using smaller centrifuge tubes?
Hello Golnaz,
I use 5mL tubes to isolate organelles and have a colleague who uses them for DRM isolation and the only times we see mixing are either when someone increases braking speed or when the machine is having a hard time maintaining temp at speed. To the best of my knowledge there is no tube size variation when performing density gradient centrifugation, and I have successfully used multiple tube sizes above and below 5mLs with little variation. The only thing I can think of would be that as the two rotors themselves are roughly 6 inches apart in max diameter. This means if you spin the roughly 10 inch SW55 Ti at the same ~23,000 RPMs it takes to generate 100,000xg on the 16" SW32 rotor your actually only hitting the mid 60,000xg range on the smaller rotor. At least thats my best estimate without breaking out the specs and doing the math. Is it possible you need to adjust for rotor diameter in your particular centrifuge?
Hi Jason, Thanks a lot for your response. I set the ultracentrifuge on 100k g and it automatically adjusts the rpm based on the rotor model (and hopefully is doing so correctly!). Do you do a shorter or longer centrifugation when you use the small tubes?
You can change the percentage of your gradient. Try using a continuous 6-20% optiprep gradient.
Hello Golnaz,
I was wondering if you already solved the problem?
Unfortunately, I encounter the same problem...
I have been the same problem. One thing that is important an can help to solve the problem is calculating the time of centrifugation based on K-Factor of rotors.
For exemple: supposing that factor K of some rotor is 44.4 and the time of centrifugation is 2 hours, if you use another one that K factor is 88.4, you have to centrifuge for 4 hours at same conditions of rpm or xg. This have helped me for while.
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