Purification of proteins: Z-alpha and Z-omega

Piotr, Emilia

1. Samples were resuspended in PBS, sonicated and centrifuged.
2. Lysis buffer was added separately to pellet and supernatant, then samples were boiled for 10 min.
3. Lysates loaded on 12% poliacrylamide gel (amount relating to 100 μl of OD=1.0 culture).
4. Gel stained with Coomassie Blue. Optimal induction conditions chosen (Fig. 1 and Fig. 2).

Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and SacI (BamHI buffer).
3. Gel electrophoresis - proper clones founded.

Cloning of truncated fragment of protein A

Michał K.

1. Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA).
2. Transformants plating on LB + kanamycin.
Z-omega induction with various IPTG concentrations
The arrow shows place of our overexpressed protein
: 1. 22°C 0,5 mM IPTG
2. 22°C 0,1 mM IPTG
3. 22°C 1 mM IPTG
4. Marker
5. negative control
6. 37°C 0,1 mM IPTG
7. 37°C 0,5 mM IPTG
8. 37°C 1 mM IPTG