His-tagged protein and SDS-PAGE migration

Richard J. Dudley rdudley at nrc.uab.edu
Thu Mar 26 12:41:20 EST 1998

James Bassuk wrote:
> wrong.  one His residue has a MW of about 100 daltons.
>         six times 100 = 600 or 0.6 kDa
> Jim Bassuk
> U Washington
> Seattle

Good math.  However, proteins aren't separated on SDS-PAGE by mass--they
are separated by charge.  SDS binds to proteins at a constant ratio
(approx. 1.4 mg SDS/mg protein), giving a constant charge:mass ratio for
most well-denatured proteins (hence the boiling step with SDS and
reducing agents).  In the classic Laemmli system, histidines carry a
positive charge, which alters the charge:mass ratio.  The extent of the
effect varies on the size of the protein involved (i.e., smaller
proteins are affected more, since there is less "regular" stuff to
dilute the discrepancy).

Now, a 64 kDa protein running at 90 kDa probably has some other issues
than merely 6 histidines--incomplete denaturation, or a very string
association with another protein (in many cases, especially with
membrane-bound receptors, proteins can remain in association even after
boiling with strong reducing agents). It's also been the case that
boiling a membrane-bound receptor can increase its apparent Mr, while
heating at 65oC for 10-15 min doesn't.


--- --- --- -- -- -- --- --- ---
* Yes, Kathy really did let me name the cat Histidine. *

Richard J. Dudley (rdudley at nrc.uab.edu)                            
Department of Neurobiology                    
University of Alabama School of Medicine       

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