help lysing E. coli to purify inclusion bodies

Ron Kagan rkagan at ewald.mbi.ucla.edu
Tue Jun 28 14:41:28 EST 1994


In article <Cs4CCt.595 at ucdavis.edu> jfh, jfhess at ucdavis.edu writes:
>Howdy all, 
>
>I (we) are making mamalian proteins in ecoli using pT7 and BL21(DE3).
>
>I've had success with the system but my lysis is always a crap shoot. 
>Sometimes lysis occurs easily sometimes not.  I have been following the
>methods in enzymology, nagai and thorsen protocol, using lysozyme,
>followed by deoxycholic, NP40, and DNAse 1.
>
>What do other people do?  Post some suggestions here or email me.
>
>
>John Hess, PhD                    Phone me 916 752 8420
>Dept of Human Anatomy             FAX me 916 752 8520
>University of Calif               Email me jfhess at ucdavis.edu
>Davis, CA                         or leave me alone, your choice.


I've been using either sonication (3X30 sec. at setting 3-4, place on ice
for 1 min. between rounds) or a French Press (15,000 psi, twice) to lyse
E. coli.  Both methods give good protein yields and active enzyme(s).  


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"My Kindness is not random, and nothing senseless was ever beautiful."

                                                 - J. Zabriskie
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Ron Kagan
rkagan at ewald.mbi.ucla.edu



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