Way back in the 1950s and 1960s, gradient density centrifugation was commonly used to isolate and characterize proteins based on size and density.  It was cumbersome, tedious, and expensive, which is why it has been replaced for the most part by electrophoretic and chromatographic techniques. I suggest you read some of the textbooks and methodology journals from that era before you decide to use preparative ultracentrifugation.   By the way, we see one carryover from that era when we talk about 16S RNA - the S stands for Svedberg, a unit used to express the coefficient of sedimentation in ultracentrifugation.

Good luck!

Way back in the 1950s and 1960s, gradient density centrifugation was commonly used to isolate and characterize proteins based on size and density.  It was cumbersome, tedious, and expensive, which is why it has been replaced for the most part by electrophoretic and chromatographic techniques. I suggest you read some of the textbooks and methodology journals from that era before you decide to use preparative ultracentrifugation.   By the way, we see one carryover from that era when we talk about 16S RNA - the S stands for Svedberg, a unit used to express the coefficient of sedimentation in ultracentrifugation.

Good luck!

hi

I think this is not feasible unless you precipitate whole proteins with ammonium chloride.

good luck

Hi Mahbobeh, I agree with Greg. Proteins have similar densities.

If you are isolating inclusion bodies (of mangled up denatured protein masses that are mostly useless for any structural or functional studies), then you can centrifuge them out.

Good luck in getting anything meaningful out of them.

Even if you do decide to use preparative density gradient centrifugation as a purification step (most useful for high molecular weight complexes and membrane purification), you are not likely to get a pure protein with a single purification step. You will probably have to use at least one more step, probably chromatography.

SDS-PAGE is almost always used as an analytical step to examine your protein for purity and to investigate subunit structure, not as a preparative step.

Thanks all for your kind recommendations.

Is there any specific reason that you want to use centrifugation? Generally I would not recommend limiting yourself to a singular type of purification. Typically you get better results from a combination of methods.  

Technically you could as Reza suggests, use ammonia precipitation as bulk method to get rid of most other proteins. If your expression is low, this may not be a good method as you will probably lose some of your protein. 

Heat-precipitation is also an option if you have stable protein.

Then if you insist on using centrifugation, there are several systems that allow you to combine gravity flow columns (ion exchange + any beads for tag-purification) with centrifugation (these are mostly for small volumes and will not give you high resolution).

I would prefer to go with a self-packed gravity flow column if other purification systems are not available.  

Potentially you could also use spin filters with different cut-offs to get rid of smaller and larger proteins (although this is not a nice method). 

Hope it helps :)