Protein but not DNA denaturant

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Tue Nov 8 10:06:47 EST 2011


Hello all,
I am working on something that may require sequential "washes" of active
enzymes over a bound DNA substrate, with inactivation of the enzymes
after each step. Preferentially, inactivation can be achieved as a rapid
"wash" rather than requiring a lengthly incubation.

Because the preferred method of DNA binding is through a 5'
cellulose-binding aptamer rather than a covalent attachment to gold or
similar, I would like to avoid conditions that could denature the DNA.
So, while heat will denature most proteins readily, it also denatures
DNA, which I am trying to avoid. The cellulose binding aptamer appears
to employ G-quadraplexes which may not refold correctly from certain
perturbations, so the less likely they are to unfold the better.

Urea appears to affect DNA melting temperature and thus probably
structure, and the same goes for Guanadine Hydrochloride, alcohols, etc.

Unless a balance of Urea/Guanidine could be found that spares my DNA
while denaturing the proteins used, I have been looking at some level of
SDS: the aptamer is resistant to at least 0.01% SDS, but I am unsure
what concentration of SDS will be needed to denature proteins?

Likewise, I'm looking at salt-precipitation of proteins, but I am unsure
of which precipitating salts are likely to affect DNA structure.

Are there any protocols out there that might be useful for this
application? A "protein-killing" buffer that is largely benign to DNA?

The proteins in question will be restriction enzymes and a polymerase,
so they are unlikely to be particularly resistant to denaturing conditions.

Thanks,
Cathal

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