expressed protein is 5-6kda bigger than

billing at vetsci.microvet.arizona.edu billing at vetsci.microvet.arizona.edu
Wed Aug 21 14:28:01 EST 1996


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


>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



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