His-tagged protein and SDS-PAGE migration

Arnoud van Vliet arnoud at vanvliet.u-net.com
Thu Mar 26 04:59:31 EST 1998


Remember that on a SDS-PAGE gel you get an approximate MW, not an
absolute MW. Depending on the sequence it can run different. I
remember an example where a change (not an addition!) of one amino
acid caused a 4 kDa change in MW. I have expressed a protein in the
Qiaexpress and the pTrcHis vectors, where the only difference is that
the pTrcHis protein has a 5 amino acid addition, the enterokinase
cleavage site. The pTrcHis protein runs at 21 kD, the Qiaexpress at 18
kD. The 6 His tail changes the protein quite a lot, so the message is:
never ever only rely on SDS-PAGE for MW!

Just my 5 cents worth...

Arnoud


On Wed, 25 Mar 1998 22:07:20 -0800, James Bassuk
<bassuk at u.washington.edu> wrote:

>
>nonsense.  his-tag won't by itself cause nothing more than an increase
>in MW of 600.
>
>but, being a recombinant protein, all kinds of screwy things can happen.
>your question is too general because every protein would have an
>emperical answer.
>
>Jim Bassuk
>U Washington
>Seattle
>
>
>On Tue, 10 Mar 1998 ct133 at columbia.edu wrote:
>
>> Hi,
>> 
>> Does anybody know whether his-tagged protein expressed from pRSET
>> vector (Invitrogen) can migrate aberrantly on SDS-PAGE? 
>> 
>> Thanks,
>> 
>> C. Tunyaplin
>> 
>> 
>




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