Prot./DNA interaction and SDS-PAGE

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Thu Oct 12 12:14:54 EST 2000


My expressed His6 DNA-binding protein shows 2 very close bands on SDS-PAGE.
Could someone share his opinion whether these 2 bands could represent the
protein in two forms, bound and non-bound to DNA? Could the binding to DNA
be retained after 5 min boiling in SDS-PAGE loading buffer.

The isolation procedure I use includes DNase treatment of the protein AFTER
binding to the IMAC resin, with subsequent elution with imidazole
(nondenaturing conditions).

There is another possible explanation for 2 bands on a gel: the expressed
gene has the second start codon after 6His, i.e., ATG-6His-ATG-gene-Stop.
However, I am not sure that the second ATG can cause expression of the
protein in 2 forms, with and without 6His. Since the isolation procedure
employs DNase treatment on the resin, there is a chance that non-His6
protein may be bound to the resin if it shares a DNA molecule with the His6
protein bound to the resin.

Any comments and suggestions would be greatly appreciated.

Emir







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