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.
Hi, we have used sonication to purify several membrane proteins also. It is not the most efficient, a french press or still better a microfluidizer are your best bets. However, if you do not have either of these the sonicator works pretty well. In those cases where we use a sonicator we a) chill the tip (this doesn't really help with the microtips but, with the larger probes the overall temperature of the solution remains significantly lower, b) use rlysozyme, we find that you need much fewer cycles to achieve lysis with this in your buffer. The more you use the easier it is to lyse but there is a cost benefit analysis you need to perform on you own. Hen egg white may also work but you need much more (its cheaper though) and then you need to get rid of it. A comment that may be applicable to you, in general, membrane proteins are reasonably stable in their membrane environment (hence the substantial stabilization of these proteins when incorporated into lipid nanodiscs, see NMR studies of Wagner and his colleagues). Therefore, as long as there is no detergent preset (most common detergents are effectively weak denaturants of the membrane protein), and the proteins is largely embedded in the membrane or not a complex involving both membrane embedded and soluble components, for the average membrane protein, sonication should not be a great problem. Further, we have found that the quality of the purified proteins is better when you isolate the membrane fraction (high speed centrifugation ~100-120k 30-60min) and do several washes before solubilizing the protein with detergent. This eliminates much of the misfolded protein.
However, all advice in general terms on purifying membrane proteins or for that matter soluble proteins should be taken with a grain of salt. There is certainly too much variability to give a pat answer and you should really investigate each case to ensure that you have optimal conditions. However, if yours is typical then you may find some of this and the above information useful.
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