QIAGEN native lysis protocol

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Jun 18 09:53:48 EST 1999


In article <7k5o9l$lb$1 at testinfo.uoguelph.ca>, kurbanic at uoguelph.ca (Kevin Urbanic) wrote:
:Hi,
:
:I am currently trying to natively purify a His-tagged protein using 
:QIAGEN Ni2+ resin.  There protocol for native cell lysis is not very 
:efficient.  Does anybody have any real good methods for natively lysing 
:E.coli cells?

Wash cells once with Tris 50 mM, 2 mM EDTA, pH 8.0, resuspend 
in 1/100 of culture volume of the same, add lysozyme to 1 mg/ml (from 100 
mg/ml stock in water), incubate on ice for > 30 min, freeze o/n at -80 
(-20 will do, too), thaw, add MgCl2 to 12 mM final, add 0.1 mg/ml DNAse, 
incubate on ice for > 30 min with frequent rigorous mixing. Centrifuge
at > 40000 g for 30 min, take super. If you care about the column to 
use then, further 30 min spin in ultracentriguge is recommended. 
Worsks great, particularly for large volumes and never heats the 
sample up like sonication and french press do. Protease inhibitors
are optional and required only if extensive proteolysis problem is 
veryfied.

        - Dima




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