Dear All
Is there anybody who has expertise in DNA binding protein purification?
I try to purify a transcription factor and few hours after purification the protein completely precipitates... Even if it is not precipitated it stays as a higher oligomer and do not even bind to the DNA.. However, when I add some TCA cycle intermediate during purification the protein takes two forms trimer and oligomer.. The trimer binds the DNA efficiently.. Can it mean that the intermediate serve as a cofactor of the protein.. Is there any other reason why the protein takes two forms in the presence of an intermediate?
It can be form of regulation - metabolite is present -> TF binds to DNA and alters transcription
Very interesting. My first thoughts go to the question of the presence or absence of Mg2+ and chelators. What is the environment in which you have your purified protein?
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