I have been doing saccharose gradient purification for a while, and a doubt has recently arisen.
I understand that for an organelle, membranous system, or solid precipitate there will be a specific "equilibrium" density during centrifugation.
On the other hand, as far as soluble molecules are concerned, I had the idea that they have the ability to move (probably slowly) by diffussion more or less freely along the gradient. A matter of time, I guess.
However, do different saccharose layer interfaces act to some extent as barriers to this diffusion, in opposition to the more "straightforward" diffusion inside one give layer?
More importantly, are soluble proteins expected to diffuse freely through a gradient, or will they focus at a given density, regardless of the fact that they are not denaturalized/precipitated?
Thanks a lot.