Cell Lysis

(of Gram Negative bacteria)

Once you’ve produced your desired protein in your microorganism, you’ll likely want to purify it for analysis, yield estimates or to actually use. It can be a delicate balancing act to be rough enough to break membranes, but not to damage your protein.
The following protocols are useful for lysing gram-negative E. coli, although they can likely be adjusted for other cell types.

Protocol: Cell Lysis by Beadbeating

This protocol requires a special piece of equipment (creatively named a beadbeater) and glass beads of varying sizes. It is a mostly mechanical lysis procedure, using physical force to break open the cells, with lysozyme to aid in breaking the peptidoglycan layer.

Equipment: Bead Beater

Protocol: Periplasmic Lysis with Chloroform

This protocol uses chloroform to dissolve the outer membrane of bacteria, without disturbing the inner membrane. It is specifically useful for purifying proteins that have been exported to the periplasmic space, such as with our Ecotin-Insulin fusion molecules. This will not work for purifying cytoplasmic protein.