expressed protein is 5-6kda bigger than

Andrew Wallace a.wallace at queens-belfast.ac.uk
Fri Aug 23 07:15:34 EST 1996


billing at vetsci.microvet.arizona.edu wrote:
> 
> One reason for this may be the fact that these proteins are run in the
> presence of SDS which binds to them so that the net charge of all the
> molecules is the same.  Hence, separation is effectively a function of
> size, not charge (they are moving through an electric field).
> 
> The average AA binds 1.4 molecules of SDS (I think that's right).  This
> means acidic AAs bind less and basic AAs (like His) bind more.  As SDS
> is a large molecule, the extra SDS molecules make the protein "heavier",
> hence, it runs slower.
> 
> This effect is a common one with basic proteins.  I can't see that it
> would be much different with recombinant 6XHis-proteins.  I don't have
> any refs, but a basic PAGE text might be a place to start.
> 
> Mind you, I could be way off base with this :-)
> 
> Steve
> 

Well, it seems like a reasonable explanation to me so I don't think you 
are off base at all.

Andrew

> >Christoph H. Winter wrote:
> >
> > >> In our workgroup this was found as well!
> > >
> > >Why?
> >
> > Sorry, I can1t explain it exactly...
> >
> > In our group it was detected on a SDS-gel with a His-tagged scFv (should
> > have 28 kD by calculation, showed ca. 33 kD) and a non-related smaller
> > protein my colleque cloned. Maybe(!) it1s caused by the positive charge
> > the His carries, retarding the whole protein...?
> >
> > I don1t know about proteins with a high amount of lysins and/or arginins,
> > maybe they behave similar.
> >
> > BTW:
> > Citation of the QIAexpressionist, Appendix B:
> > 3Some proteins with 6xHis tags attached run more slowly on SDS gels than
> > equivalent untagged proteins, and may appear to be several kD larger than
> > expected2
> >
> > Bye
> >
> > Christoph

-- 
======================================================================
 Andrew Wallace,Ph.D., Queen's University Belfast,  N. Ireland (UK)   
 a.wallace at qub.ac.uk   http://www.qub.ac.uk/b&bchem/awpage/wallace.htm   
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