Proteins which run high on SDS-PAGE

Achim Recktenwald achim at ibex.ca
Tue May 14 18:54:58 EST 1996


RHODES at UCONNVM.UCONN.EDU wrote:
> 
> On Wed, 8 May 1996 13:15:56 GMT Sam Simons said:
> >Prolines introduce natural "kinks" in the protein which aren't
> >straightened out by denaturing. Since denaturing in SDS creates linear
> >polypeptides, this bending will give a larger stokes radius to the
> >protein which will cause it to run slower through SDS-PAGE.
> -------8<------(snip)
> Would you like to elaborate on that?  It seems contrary to what I recall
> about f for long rods as opposed to ellipsoids or spheres.  Isn't the
> rod an 'extreme' case, such that any alteration of the shape would
> decrease f?  As I suggested to the original poster, the possibility of
> charge anomaly (i.e. extra +'s) is far more likely, and would not require
> something completely out of line with the size range we're dealing with
> in this case.  Nevertheless, If someone can show that
> |
> |
> |          moves more slowly than  ___________________, I'd like to see it.
> |          I'd be _really_ surprised (not the first time) to see that
> |________          _   _
>             ______| |_| |____   would run more slowly.
>                              |
>                              |
> 
> |                              O==O                            |
> |  DAVID G. RHODES             O==O  PHONE 860-486-5413        |
> |  SCHOOL OF PHARMACY; U-92    O==O  FAX   860-486-4998        |
> |  UNIVERSITY OF CONNECTICUT   O==O                            |
> |  STORRS, CT  06269-2092      O==O  RHODES at UCONNVM.UCONN.EDU  |
> |                              O==O                            |


The average protein binds 1.4g SDS/g protein. The charge of  the high number of SDS 
molecules bound to the protein should more than outnumber any intrinsic charge(s) of 
the protein. Glycoproteins bind less SDS and, therefore, run at higher molecular 
weights.

To properly saturate the protein with SDS, it should be denatured by incubation at  
high temperatures in the presence of SDS; therefore the usual heating of the sample in 
SDS-sample buffer.
Though I am not quite sure how, the prolines could somehow 'inhibit' the denaturing of 
the protein upon heating and diminish the number of SDS-molecules bound. The effect 
would, as it is the case with glycoproteins, lead to an apparent higher molecular 
weight.


Achim



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