Cell disruption without sonication - (Jul/17/2004 )

Hello everybody,

Help !!!!

I am expressing my protein in E. coli BL21, and I am looking for another way to disrupt the cells without sonication… Does anyone have a protocol ?!?!?! I tried lyzosyme followed by frezze-defrezze methodology, but the separation of pellet, supernatant and debrit was terrible… I needed to centrifugate several times, so I am not sure about the integrity of my protein… I am growing a culture of 50 mL.

Please, send me some advice !!! Thanks in advance,

Claire

Try BugBuster protein extraction reagent from Novagen

for the gentle disruption of the cell wall of E. coli, liberating soluble protein. It provides a simple, rapid, low-cost alternative to mechanical methods such as French Press or sonication for releasing expressed target protein in preparation for purification or other applications. The proprietary formulation utilizes a detergent mix that is capable of cell wall perforation without denaturing soluble protein.

How about French Press? What is your objective in avoiding sonication?

Hi !!!!

Thanks for your posts !!!!

I just saw this BugBuster reagent from Novagen, I wrote them yesterday asking about price and deliver. I am writing from Brazil, and I didn´t find this information in their webpage.

Our sonicator isn´t available right now, so I am trying to figure another away of doing it... I don´t have a protocol of French Press using, would have one to send me ?

Hope to hear from you soon, and thanks a lot for your help !!!!

Claire

Claire

If your lab has problems with reagent procurement because you are in Brazil; try doing a simple freeze-thaw. Freeze pellet suspension under liquid nitrogen, thaw at 37C, repeat 2-3 times. Works for only some bacteria like E coli and salmonella.