Hello,
I would like to use Benzonase in my lysis buffer to reduce viscosity. I have been using RIPA buffer (50mM Tris, 150mM NaCl, 0.5% Na deoxycholate, 1% NP-40, 0.1% SDS, pH 8.0) to lyse my cells, as I wish to lyse the nuclear membrane.
However, it seems that the amounts of sodium deoxycholate and SDS I am using will have a high impact on Benzonase activity. From their literature, the maximum recommended SDS concentration for use with Benzonase is 0.05%, and the maximum sodium deoxycholate is 0.2%. Would those concentrations be enough to lyse the nuclei?
If not, can someone recommend a good lysis buffer to use with Benzonase? I do notice that Benzonase apparently works great with Trition X-100 concentrations of 1%, which is much higher than typically used in buffers which spare the nuclei.
Thank you!
Have you experimentally verified that your benzonase really doesn't work with the RIPA buffer? I haven't done any extensive tests, but I did use Benzonase in lysis buffers with as high concentrations of SDS as 1%, and it still did its job. Higher concentration of the enzyme and longer incubation may be required, but you should be able to easily test that.
If your IP buffer contains less and weaker detergents as literature mentioned, it should work. Bivalent ions is required for DNase. And I don't believe 1% SDS is compatible for Benzonase, unless you overdose it, maybe it can survive for a few minutes.
Benzonase works good in RIPA buffer. It clearly reduce the viscosity.
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