I have been trying to use a CsCl gradient to purify an intracellular virus. However after centrifuging at 100000xg for over 20 hours there are no bands and the cellular proteins are distributed uniformly throughout the fractions. Does anyone have any idea why this is happening? Thanks
.. what type of rotor do you use; can you please give details on CsCl concentration, buffer and temperature?
Thanks for your response.
I used a final concentration of 0.4g/ml of CsCl, the sample had been dialysed into 20mM Tris buffer. I use a SW 32 Ti rotor and the centrifugation temperature was 10C
Sounds a bit as if your protocol has initially been made intitially for pre-formed step gradients. For gradients to be formed during centrifugation, centrifugation time is rather short.
Maybe, you have a look at http://www.virusecology.org/MOVE/Method%2011.html - There, a good protocol for step gradients is described.
Good luck! - Johannes.
Thanks alot, I will defenitely try that.
Also, how long do think it will take for a gradient to be formed during centrifugation?
I think your protein may be breaking down in CsCl. You can try CsSo4., if not ficoll or sucrose.
Thanks everyone
I dont think my protein is breaking down, because in the SDS Page I see a uniform distribution of all proteins, not just mine
... I recommend to use pre-formed step gradients. For complete phages, the CsCl concentration is much lower than for isolation of DNA. Thus, gradient formation takes even more time than for DNA isolation. At the relatively low g-force in you rotor, you could easily end up with 60 hours (at least 40). Step gradients are easy to do, and you have the result normally over night.
Just try, it's not complicated.
The rotors to use for self-forming CsCl gradients are shallow fixed angle, or vertical rotors or near vertical rotors. NEVER use a swing-out rotor for self-forming gradients of caesium salts as the gradients take ages to form and crystallisation may cause rotor failure. Given the conditions you quote in my experience a gradient MUST form if there is no gradient when analysed then it has been disrupted by rotor instability or very fast deceleration
Just to add that the time for the formation of an equilibrium gradient is proportional to the square of the sedimentation distance!! Double the distance then the time required is four times longer
Preformed CsCl gradients are very unstable because of their low viscosity and CsCl does inactivate many viruses. Most modern protocols now use Optiprep a non-ionic iodinated density gradient medium which gives much better results than CsCl gradients.
Dear Sir,
I wanted to separate 5nm sized particle and 200 nm sized particle. should i go for CsCl centrifugation. what should be the concentrations to be used.
To separate particles based on size, you would need to use a sucrose gradient. CsCl gradients separate molecules based on their density.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology