Dear all,
I need to purify anoxically a protein with Fe-S clusters (sensitive to oxygen). I used to do cell lysis using a French press but because the instrument is too big to be place in my anoxic chamber, I was considering to lyse the cells by sonication. Anybody knows whether sonication could affect the binding of Fe-S clusters with the enzyme, and subsequently affect its activity?
Thank you very much for your help,
Solenne
Is it reasonable and applicable to use bead beater homogenization units instead of french press homogenizer or adding antioxidants to your lysis buffer to inhibit the oxidation? For sure You can use a sonication probe to lysis but being sure to prevent clusters from oxygen exposure, nitrogen purging before the application may be beneficial.
Rapid and multiple freeze-thaw cycles as the traditional method can also lyse the cells in some cases.
It depends on the volumes of the extracts you want to prepare. For very small amounts of cells (2-3g) you could use Bugbuster inside glove boxes. The lysis is not as efficient as physical methods but good enough for breaking the vast majority of cells and the big advantage is that the lysis is mild. For larger volumes (>100ml) a French press is much better, what can be done is to set gas lines and closed bottles with tubings that make the "bottle-frenchpress system" anaerobic.
Fe-S clusters could fall apart with sonication, but it depends on how buried or stable is the cluster inside your protein. But you could try using the sonication as suggested above, it is very simple and quite fast for small volumes and you can check the result quite fast by just looking at the color of the purified protein, UV-vis, BCA and Fe measurements.
- Badges
- Science topic
- Similar topics
- Instrumentation Engineering
- Instruments