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.