I follow a protocol to get protein from E.coli cells after sonication.

I used to grow 6 litres of large cultures and add IPTG to express the protein. I centrifuge for 10 mins at 8,000 rpm and get the pellets. Then I do sonication and centrifuge again at 20,000 for 1 hr.

My question is: can I do the last step of centrifugation at 4000 rpm for 2 hr? The centrifuge is no longer working at high speed (20,000rpm)


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    $\begingroup$ It is better to indicate centrifugation speeds in terms of g instead of RPM. We basically want to know the centrifugal force acting on the sample. The RPM required for a particular amount of centrifugal force depends on the rotor radius. $\endgroup$ – WYSIWYG Nov 27 '18 at 12:29

No, if you use only centrifugation to separate proteins from other parts of the (bacterial) cell, you need very high centrifugal forces. The actual speed (in rpm) you need to separate soluble proteins from the rest of the cell is mostly dependent on the rotor of the centrifuge, because both together determine the acceleration you get, which should be in the order of at least 10,000 - 100,000 g.

If you do not have a (working) ultracentrifuge available, you could try to precipitate your proteins from the lysate, which would allow you to use a normal centrifuge to collect it. However, this won't be an option if your protein can't be properly re-folded and you need it functional.


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