Prot./DNA interaction and SDS-PAGE

Michael L. Sullivan mlsulliv at
Fri Oct 13 13:20:37 EST 2000

>""Michael L. Sullivan"" <mlsulliv at> wrote in message
>> Another possibility for seeing the two forms after IMAC (assuming they are
>> his/not his tagged versions) is that the protein forms heteromultimers.
>> Mike
>I did not get the issue of heteromultimers. You mean binging of His/nonHis
>to each other? Then boiling in the loading buffer would destroy these

I don't have your original post any longer, but my understanding is that
you loaded an E. coli extract expressing your protein on an IMAC column
under non-denaturing conditions, DNase treated, then ran what you eluted on
SDS-PAGE.  You suggested perhaps the two forms were his-tagged and
non-his-tagged, as there was a second ATG past the his-tag from which
translation could initiate.  What I am suggesting is that heteromultimers
might have existed in your original extract (which was made under native
conditions, righ?).  If a heteromultimer contained one his-tagged protein,
the whole multimer would purify.  When you ran the eluted material on
SDS-PAGE, the two forms, his-tagged and non-his tagged might resolve into
two bands since a his-tag can change the apparent molecular weight much
more than might be expected (i.e. several kDa rather than the expected ~0.6
or so) .

Did I misunderstand or misread your original post, or is what I suggest a

If you have an anti his-tag monoclonal Ab, you could see if either or both
bands have the his tag.

Hope this helps.


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


More information about the Methods mailing list