Sedimentation velocity

Go to the profile of nekameneka
Sep 01, 2017
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Hello everyone,

Is there someone who has experience in sedimentation velocity experiments? I would like to know how to set the parameters for the protein of interest, how do I determine the speed and time in this experiment...
Also, anyone experienced in Virtual Cell software??

Thank you!

7 Comments

Go to the profile of mdfenko
mdfenko 5 months ago

it's been a very long time since i worked with sedimentation coefficients (used for pelleting viruses).

the formulae can be found in the manual from the ultracentrifuge (or the tubes catalog, i don't remember which), at least that from beckman-coulter.

determining sedimentation coefficient requires a series of spins with various concentrations of sample and knowledge of the viscosity and density of the medium through which you sediment.

you can get a decent estimate if you run density gradients, such as with cesium chloride (which creates the gradient during the spin) or sucrose or glycerol (you pour these gradients).

if you have an analytical ultracentrifuge, then you spin through the gradient and it will give you data to determine the coefficient.

sorry if this isn't too helpful, i could write the formulae but they may not make sense without the context of the manual (which i don't currently have at hand).

Go to the profile of nekameneka
nekameneka 5 months ago

Dear mdfenko,

Thank you for the reply. Yes, I would like to do it in sucrose gradient. I have Beckman Coulter Optima ultracentrifuge. I will search for the manual and check it.
I am sure I will have questions so I'll be back later!

Go to the profile of nekameneka
nekameneka 5 months ago

Dear mdfenko,

Here I am back with questions about sedimentation velocity. Maybe you can help me understand these formulas.
I found this in the manual for Hitachi ultracentrifuge (from page 9 to 10 they give formulas):
http://centrifuges.hitachi-koki.com/rotor/ultra/s99920413.pdf

Please if you have time, can you explain to me more, I do not really fully understand.
To give some kind of idea why I want to use this experiment: I want to determine the stoichiometry of 2 proteins where I think one protein is nucleator of another.

Thanks a lot in advance!

Go to the profile of mdfenko
mdfenko 5 months ago

are you combining 2 proteins then trying to separate any complexes formed from the remainder of the starting proteins?

if that's the case then if you must use centrifugation to separate them, you can do that with a density gradient (glycerol or sucrose) in which the three proteins (2 starting proteins and complex) will separate but remain suspended. you not only don't need to pellet the complex (which is what the equations help you to do) but it may even damage the complex. plus, if you spin too long then some of the starting proteins may also pellet, throwing off the stoichiometry.

personally, i would separate them with sec.

Go to the profile of nekameneka
nekameneka 5 months ago

Dear mdfenko,

No, that is not what I am trying to do. Sorry I was not very clear, here I send you a paper which finds leiomodin is an actin nucleator, please if you have time read about figure 4 (C and D are sedimentation velocity results). I would like to do this kind of experiment.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845909/pdf/nihms187505.pdf

Thanks a lot.

Go to the profile of mdfenko
mdfenko 5 months ago

apparently, the authors of the article got their values, not through use of the equations, but, directly from the analytical ultracentrifuge's analysis package.

do you have access to an analytical ultracentrifuge (or a preparative ultracentrifuge with the analytical optics package)? that will be your best bet to duplicate the authors' procedure.

Go to the profile of nekameneka
nekameneka 5 months ago

Oh I see, hmmm yeah I think I can find one. Thanks a lot for the help!