I assume that you are asking about proteins. In native gels,
proteins migrate according to their size & shape (Stokes radius - the
radius of the sphere described as the protein tumbles randomly in solution)
and their intrinsic charge. In the standard Tris/gly system, in the
absence of SDS, proteins which have a negative charge will migrate into the
gel but those which are neutral or positively charged at the pH of the
sample buffer will not bee seen as they will either not migrate out of the
well or will move towards the cathode - an important point to bear in mind.
In some flat-bed systems where the sample is loaded to the surface of the
gel (e.g Phast or equivalent), loading the sample to the centre of the gel
can allow visualisation of acidic and basic proteins in the same run.
Highly oligomeric proteins may have trouble entering a gel if they are
several hundred kDa big as the assembled form. In these circumstances,
non-polyacrylamide gels (e.g. agarose) may have to be used. Some workers
use polyacrylamide gels with a transverse gradient of urea to examine the
stability of oligomeric structures or folding units within monomers. By
applying a sample across the width of a gel, its migration rate will change
in response to increasing urea concentrations as an oligomer dissociates or
a monomer unfolds, so changing the Stokes radius while not necessarily
altering the charge state. Native gels can be very useful things! With
careful calibration, native gels can be used to determine the mass of an
oligomer (Ferguson plots).
In the presence of SDS, the detergent helps unfold the protein and binds at
a constant amount per unit mass for most proteins; this is one molecule for
every 2-3 amino acid residues if memory serves me correctly. Thus proteins
have a constant charge/mass ratio and migration in the gel becomes
dependent on mass alone, so an SDS system is ideally suited for mass
determination. There are exceptions though, where the structure of a
protein influences (reduces) the binding of SDS (e.g. poly-Ala stretches,
glycosylation, some post translational modifications) and in these
instances, proteins migrate at apparently higher mass than expected.
Len
>Hi everyone,
>
>What is the mechanism of the separation for a native gel? Thanks.
>
>
>
>Bob Lee, UIC
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Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
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