Protein but not DNA denaturant

AllisonH via methods%40net.bio.net (by nospam from gmail.com)
Tue Nov 8 17:54:24 EST 2011


Could you play with pH?  For example, low pH is used to elute antibodies 
off a protein A column, and the eluate needs to be neutralized right 
away so that the antibodies aren't denatured....

It's been a while since I worked with DNA so forgive me if this is 
completely crazy.

Allison


On 08/11/2011 10:06 AM, Cathal Garvey wrote:
> 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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