I loaded my yeast cell extract, treated with cycloheximide, onto a sucrose gradient. After ultracentrifuging the samples, the UV detector stopped working before I could do the fractionation, so I decided to freeze the tubes at -20 degrees. I still haven't thawed this samples. Has anyone got an idea if I would still be able to see a good profile? Any advice on how is best to thaw the tubes with the gradient? In ice, or at 4 degrees?
We have not done this, but I would say that the gradients are fairly robust. Colleagues say they have stored post-centrifugation gradients at 4°C overnight before and got a normal profile, or left samples in the centrifuge rotor overnight and got a normal profile the next day. Assuming all you want is a A254 trace I would think thawing at 4C or room temperature will both be fine. If you want to collect fractions to analyse proteins or RNAs then probably best to slow thaw in a fridge or cold room for several hours.
Maria,
In my opinion the gradients are robust per Graham's discussion. However, poly somes are membrane based entities and will be disrupted by freezing.
Thank you for both your answers. Finally, I decided to thaw at 4C, which took less than four hours, and check the profile. But it was quite bad: I got a wide RNP peak, but then no more clear peaks at all. I suppose it was the effect of freeze-thawing, so next time I have to postpone the profiling I will store the gradient at 4C without freezing, according to both your suggestions. Thanks again
Maria Teresa Martinez-Pastor , thank you so much for the feedback which can help a lot to other people.
- Badges
- Science topic
- Similar topics
- Computer Science
- Program