I'm trying to purify membrane protein that is expressed in E. Coli.
I have read several papers about lysing E. Coli Cells to purify target membrane protein.
And found most of the researchers use French press or Microfluidizer.
However we don't have those devices and we have only sonicator.
My questions is why sonication is not good for lysing E. Coli to purify membrane protein.
And if there is someone who use sonication, please tell me the condition.
I think the main reason that most people do not use sonication as a lysis method when purifying membrane proteins is due to possible heat problems. Usually the local temperature around the probe can get relatively high, even though the solution is stirred and on ice. This is normally not a big problem for soluble proteins, but membrane proteins are as you know usually a bit more sensitive.
However, it can still definitely be used, also for membrane proteins. Just make sure you cool the solution the best you can, and have relatively long time breaks between the pulses. How well it will work is obviously dependant on how sensitive the membrane protein in question is.. but you wont know until you try.
Good luck!
Hi There,
My advice is NOT to use sonication, because "it turns the membrane inside-out and as such destroys the structural integrity of the membrane and the proteins associated with it.
Kind regards
Mansi
In our lab we purified a potassium channel from E.coli using sonication. The protocol implies 5 cycles of sonication on an ice bath using a Branson probe-type apparatus, with small tip and increase in potency from 3 to 7. Each cycle takes 60 sec and intervals of 60 sec between cycles. The final protein sample can be purified, reconstituted and is fully functional.
Good luck!
I've purified a few hundred different membrane proteins expressed in E Coli, lysed by sonicaton. Followed by extraction in DDM. Seemed to end up with a good percentage of 'happy' proteins to work with.
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