Bacteria cell lysis

Richard_Heath heath at mbcf.stjude.org
Tue Feb 28 12:24:04 EST 1995


I use the BL21(DE3) strain, and find that the Triton-X100 procedure given in
Current Protocols works very well ... I know you don't want to add lysozyme,
but I add it to a final of 0.1 mg/ml a couple of minutes before adding the
triton... No obvious band on a Comassie-blue SDS gel, and it doesn't bind to
the Ni resin.

To check if your protein is made insolubly, as one respondant suggested, gel
samples of total cell protein (just boil cell pellet in SDS-loading buffer)
before and after induction....

My 2 cents worth, Richard
 

In article <bcarey-2202951920560001 at e_dickenson.zoo.uga.edu>, bcarey at zookeeper.zoo.uga.edu (Brian Carey) writes:
> Dear Netter, I discovered tonight that I can't lyse bactera. Would
> appreciate all suggestions. Here's my story. The cells are BL21(DE3).
> Cells from 200 ml culture OD600=1.0 was suspended in 5 ml of high salt
> lysis buffer (no lysozyme), freezed/thawed 2x before and 2X after 10
> cycles of 30 sec sonication at max setting. The pellet I got after spining
> was full of unlysed cells. I thought maybe the whole mass was too
> concentrated. So I resuspended the cells again, added another 5 ml of
> buffer, sonicated 20 X 30sec and spin. I still got a huge pellet of
> unlysed bacteria cells. I'm trying to purify over-expressed protein in
> these bugs and would like to avoid the use of lysozyme if possible.
> Any idea?
> Thanks in advance.
> 
> Tari
> University of Georgia 
> Cellular Biology Dept.
> E-mail: wll at zookeeper.zoo.uga.edu



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