Sucrose gradient purification - protein molecular behaviour

[karel5_98]

Hi everyone.

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.
Regards,

Karel

[mchlbrmn]

I don't know the specifics of your gradients, and what densities proteins enjoy floating at, but being dissolved will not stop them. I do have experince with purifying plasmid DNA, definitely dissolved, in a dense cesium chloride salt gradient. If you have a step gradient (not an incremental gradient like I used to make by spinning a dense CsCl solution overnight) I wouldn't imagine that an interface would keep a dissolved protein from finding it's density unless something else accumulated at the interface stopped it (but this is my sense, not real knowledge).

[relaxin]

When I was a graduate student, we determined molecular weight of a soluble enzyme using sucrose gradient centrifugation in a rotor that allowed monitoring the migration of the protein as it spins. Yes, soluble protein migrates down a sucrose gradient as a specific band under the centrifugal force. The key is to get the right sucrose gradient. I guess you will need higher percentage sucrose than those used in organelle purification.