Prot./DNA interaction and SDS-PAGE

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Oct 13 09:05:27 EST 2000


Actually, I think it's pretty well documented that 6 histadines *can* make
a big difference in apparent size on SDS-PAGE (I've read of researchers
claiming to have seen up to 15-30 kD shifts).

Another possibility for seeing the two forms after IMAC (assuming they are
his/not his tagged versions) is that the protein forms heteromultimers.

Mike

>Hi Emir,
>SDS-PAGE will definitely destroy any protein-DNA interaction, unless the
>binding is covalent.
>6 histidines is about 0.6 kDa, you won't separate such a small size dif
>on a gel. So you probably have two proteins. Unless you use a
>high-percentage gel with lots of bisacrylamide, then you may pick up
>different phosphorylation states of your protein.
>
>To be really sure, cut out the two protein bands, cut with trypsin, run
>on a high-percentage gel (or better, a tricine gel) and see if the
>patterns are the same or not.
>
>Good luck,
>Rogier
>
>Rogier Stuger
>Dept MicFizz, Free U of A
>E rogier AT biogate DOT com (spammers will be killed)
>do NOT use the my-deja.com address. I never read that one
>
>> His6 DNA-binding protein shows 2 very close bands on SDS-PAGE.
>> two forms, bound and non-bound to DNA? binding to DNA
>> retained after boiling in SDS-PAGE buffer.expressed
>> gene has second start codon after 6His
>> Emir
>
>
>Sent via Deja.com http://www.deja.com/
>Before you buy.


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


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