Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)

Piotr

Test was conducted in E.coli Rosetta strain expressing OmpA_omega_A_alpha (with and without induction) and OmpA_A_alpha.

1. Spinnign.
2. Suspending.
3. Adding of lysis buffer.
4. Boiling.
5. Putting into poliacrylamide gel.
6. Transfer onto nitrocellulose.
7. Blocking.
8. Anti-A antibody binding.
9. Washing.
10. Anti-rabbit antibody binding.
11. Developing with BCIP and NBT.

We didn't observe differences in espression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by factor other than Lon and OmpT proteases.

Purification of proteins: Z-alpha and Z-omega

Emilia

1. Bacteria centrifuged and sonicated (according to protocol).
2. Sonicate centrifuged.
3. Supernatant discarded, pellet resuspended in ddH20, centrifuged and again resuspended in ddH20.