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, [email protected] wrote: > Send Methods mailing list submissions to > [email protected] > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > [email protected] > > You can reach the person managing the list at > [email protected] > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > Today's Topics: > > 1. E. coli lysis protocol (Theresa H) > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 11 Apr 2014 22:19:56 +0000 > From: Theresa H <[email protected]> > Subject: E. coli lysis protocol > To: "[email protected]" <[email protected]> > Message-ID: <[email protected]> > 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. > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 106, Issue 7 > *************************************** _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
