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Methods Digest, Vol 106, Issue 7

Gerchman via methods%40net.bio.net (by Gerchman from research.haifa.ac.il)
Tue Apr 15 15:13:59 EST 2014


 

Greetings Theresa 

First, how do you know the cells are unbroken?
did you look under the microscope or assume that if they are in the
pellet after centrifugation they are unbroken (which very much depends
on your centrifugation conditions)? From my experience E. coli break
down very easily after lysozyme treatment... 

On a related issue, are
you sure your protein is not in inclusion bodies? Those tend to
precipitate with unbroken cells. 

Things you can try: 

Do membrane
preparation before extraction - in my hands "French Press" was much more
effective then sonication for large quantity of cells (you can also try
"Yeda Press" after lysozyme) . See full protocol bellow. 

After you get
membranes, dissolve them in buffer containing a detergent (we used 60 mM
choline chloride, 4.5 mM Tris-Cl, pH.8, 110 mM sucrose, 20% glycerol,
100 mM MOPS, pH 7, and 1% _n_-dodecyl β-D-maltoside).

Also see the
supplementary data here and the references there


http://www.nature.com/nature/journal/v435/n7046/full/nature03692.html


 Yoram 

On 2014-04-12 20:03, methods-request from oat.bio.indiana.edu
wrote: 

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> 1. E. coli lysis protocol (Theresa
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> Message: 1
> Date: Fri, 11 Apr 2014 22:19:56 +0000
> From: Theresa H
<theresahsu8 from live.com>
> Subject: E. coli lysis protocol
> To:
"methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
>
Message-ID: <DUB124-W4615BD0CF03F67F0D786289540 from phx.gbl>
> Content-Type:
text/plain; charset="iso-8859-1"
> 
> Dear forum members
> 
> I am
trying to lyse frozen E. coli cells expressing a membrane protein with
GFP fusion. After four hours in a low ionic strength lysis buffer 0.01 M
Tris, 0.03 M NaCl, lysozyme and 2 mM EDTA followed by sonication (20
minutes, 40% power), there is a still a large amount of unbroken cells
that are fluorescent.
> 
> Is there a better way for doing lysis? I am
only getting about 10 mg protein from 40 g of cells, so I am looking for
a scalable protocol. There is no Emulsiflex type homogenizer in our lab
and using BugBuster type of lysis will interfere with the detergent that
I am using to solubilize the membrane.
> 
> Thank you.
> 
>
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End of Methods Digest, Vol 106, Issue 7
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