8 Nov 2011 16:06
Protein but not DNA denaturant
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.(Continue reading)
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