Team:Cambridge/Protocols/Inclusion Body Prep

Inclusion Body Prep

This protocol was used when reflectin was expressed in inclusion bodies in E. coli, adapted from [http://tools.invitrogen.com/content/sfs/manuals/xprpur_man.pdf here]. The resulting lysate contains reflectin in solution, and allows cellular debris from the bacteria to be discarded.

Theory

Bacterial cells are lysed using sonication and a high concentration of guanidine in GLB buffer. This chemical also denatures reflectin, solubilising it so that it remains suspended in the lysate while cellular debris is pelleted and hence removed.

Practice

1. Make 8ml Guanidinium Lysis Buffer (GLB) via the appropriate protocol.
2. Equilibrate the GLB to 37°C.
3. Harvest bacterial cells from a 50 ml culture by centrifugation at 3,000 x g for 5 minutes.
4. Resuspend the cell pellet in the GLB.
5. Slowly rock the cells for 5–10 minutes at room temperature to ensure thorough cell lysis.
6. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.
7. Centrifuge the lysate at 3,000 x g for 15 minutes to pellet the cellular debris.
8. Transfer the supernatant lysate to a fresh tube. Store on ice or at -20°C.

Safety

All safety measures relating to guanidine hydrochloride in solution from the buffer protocol apply here when dealing with GLB. In addition, all consumables and liquid waste that may have come into contact with the bacteria must be autoclaved before disposal.

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