Simple question regarding protein separation...

[Jonsy123]

I would like to know how come when we separate proteins in velocity sedimentation centrifugation (i.e using a sucrose gradient), larger proteins move faster than smaller proteins, but, if you separate proteins in SDS-PAGE, larger proteins move slower than smaller proteins.

 

If we would have tried to separate proteins which were treated with SDS, by velocity sedimentation, would the larger protein move slower than the smaller, like in SDS-PAGE ?.

 

My hunch is that if you talk about globular proteins, then the larger the protein, the faster it goes through the gel, but if you talk about filamentous proteins (SDS causes all proteins to become that way), then the larger the protein, the slower it goes through the gel, because friction now plays a much bigger part. I'm not sure if I'm right though...

[Jonsy123]

Was my question not clear enough ?, or no one knows the answer ?.

[CharonY]

I would like to know how come when we separate proteins in velocity sedimentation centrifugation (i.e using a sucrose gradient), larger proteins move faster than smaller proteins, but, if you separate proteins in SDS-PAGE, larger proteins move slower than smaller proteins.

 

If we would have tried to separate proteins which were treated with SDS, by velocity sedimentation, would the larger protein move slower than the smaller, like in SDS-PAGE ?.

 

My hunch is that if you talk about globular proteins, then the larger the protein, the faster it goes through the gel, but if you talk about filamentous proteins (SDS causes all proteins to become that way), then the larger the protein, the slower it goes through the gel, because friction now plays a much bigger part. I'm not sure if I'm right though...

 

In principle you are not wrong, though you compare apples with oranges in some parts.

The point is that with each method depends on a different property of the protein. In SDS-Pages the size of the protein determines the runtime and with SDS (in addition to adding the needed charge) the proteins are denatured. However you do not really measure the mass, but just compare the run time of the given proteins with markers, which might or might not behave as the analyte.

In contrast, the analytical ultracentrifugation allows direct calcualtion of the mass. For instance with sedimentation equilibrium one can directly determine the buoyant molecular mass. Adding SDS here will mainly will directly lead to an increase in the mass of the protein. The shape usually does not an high impact on subsequent calculations here.

[Jonsy123]

CharonY,

 

Thank you very much for your answer.

 

Yes, I understand that Centrifugation and SDS-PAGE seperate proteins using somewhat different properties of the proteins (while SDS-PAGE seperates by molecular mass, Velocity Centrifugation separates by molecular mass and shape).

 

The main thing I was confused about, is why in SDS-PAGE larger proteins move more slowly, while in centrifugation they move more quickly. So, I understand that you agree that this difference is because of the usage of SDS, which open the structure of the protein, and make friction a much larger issue (in comparison to when the protein is in its native fold).

[CharonY]

Actually no. The answer to your question is the first part, both methods use different properties, regardless of detergent. SDS PAGE does not separate by mass, but by its form which becomes roughly porpotionate to its mass due to denaturation.

 

You can also seperate native proteins (as in blue native gels), and you will also find that larger proteins tend to run slower than smaller ones. Here however globular proteins will tend to be faster than linear ones with the same mass. In PAGEs proteins are retained by the pores (thus larger proteins are slower), whereas during equilibrium centrifugation they are moved towards the bottom until stopped by difussion forces. Proteins of a higher mass require higher diffusion forces to move up again (or rather have lower diffusion forces counteracting the centrifugal forces) and thus penetrate deeper into the centrifugation vial.