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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