Hello
I am struggling to do membrane protein purification for a while now. I am thinking that sonication might be a problem, the sonicator we have isn't great. Instead I tried re suspending it in lysis buffer with 0.5mg/mL lysozyme powder and incubating it.
I don't have access to a French press or cell disruptor
Is there any other ways to lysis cells?
Hi Eleftheria, i had some trubles whit sonication too. Some tricks i've found useful are adding detergent to the lysis buffer, specifically Triton X-100 at 0.2% plus lysozyme at 1mg/mL. Washing the cells with deionized water also helps (resuspending the cell pellet in water and incubating it in ice for ~10mins before collecting the pellet again, then resuspend it in lysis buffer).
After the pellet is resuspended in buffer+lysozyme+detergent I incubate it at 4 °C and 40 rpm (vortex for 1 min each 30 mins) for 2-3 hours, then follows sonication, I do 40 mins (30 s of pulse and 30s to rest). The more time you let it incubate the better results you'll have. It is a long process but its quite helpfull when one does not have the best equipment. Good luck!!
If you are using E. coli as a host for protein expression, try to use any E. coli strain as (DE3)pLysS. After expression/induction, harvest the cells and freeze the cell pellet at -80C. Next day, thaw the cells and re-do the sonication as usual, then you will get much better cell lysis.
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