Sucrose density gradient centrifugation.

Asked:
I do virus purification, first with super-speed centrifugation, and then with sucrose density gradient centrifugation, help master with sucrose density gradient centrifugation speed and speed when speeding centrifugation is linked, is equal or sucrose density gradient centrifugal speed is higher than super-speed centrifugal speed? If this is the latter, then how does it determine the speed at which the sucrose density gradient centrifuges? Thank you!
:
1, the purification of the virus, first with speed centrifugation, you should check the data to see how much speed can centrifuge the virus down. Then the speed of your step should be at least not less than that speed, your centrifugal precipitation contains viruses and protein components.
2, so your second step is to separate the virus from other proteins and impurities, and density gradient centrifugation can do that. At this time, your centrifugal speed should take into account the size of the virus and the density of sucrose, so that the virus can not precipitate the bottom of the tube and can be separated from the protein layer. This may also require specific analysis of specific issues.
asked:
thank the director for his guiding, my virus diameter is 100nm. Looked up the observational data, super-speed centrifugation is 21000rpm, 45min, I use 24000rpm2hr, I use 20%-60% continuous sucrose density gradient centrifugation, the virus floating density in sucrose is 1.18mg/ml, what is my sugar density gradient centrifugation speed? Thank you!
:
the purification of IBV (80-100nm) in our laboratory is: l virus purification enrichment (method i)
1, frozen urine sacs thawed, 4 degrees C, 11000rpm centrifugation 20min.
2, take it clear, add 30% PEG6000 (including 7.85% NaCl) to the precipitation appear (final concentration is about 7 to 8%), 4 degrees C placed (or stirred) overnight.
3, 4 degrees C, 19000rpm centrifugation 20min, discarded, precipitation with the appropriate amount of TEN dissolved.
4, 4 degrees C, 35000rpm centrifugation 3hrs, discarded on the clear, precipitation with the appropriate amount of TEN dissolved.
5, 4 degrees C, 33000rpm sucrose density gradient centrifugation 2.5hrs (sucrose gradient: 20%, 30%, 40%, 50%, 60%).
6, extract 40% sucrose belt (including IBV virus), -40 degrees C to save.
about the same as I think of you!
:
Your virus has a floating density of 1.18 mg/ml in sucrose, and if you go through a long enough centrifuge, it precipitates in this layer, which is about 40%-50% closer to 40%. (40% sucrose at 25 degrees at 1.176, 50% sucrose at 25 degrees at 1.230) now you must know the virus's seation coefficient to calculate the time she needs. Simply answering your centrifugal speed is pointless and must be combined with time to illustrate your centrifuge. A certain speed plus a certain amount of time to separate the virus.
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