Team:Warsaw/Calendar-Main/3 September 2008

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Purification of proteins: A-alpha

Piotr, Emilia

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

Fig. 1. Supernatants from A-alpha induction with various IPTG concentrations The arrow shows place of our overexpressed protein: 1. 22°C 0.5 mM IPTG 5 h 2. Marker 3. 22°C 0.5 mM IPTG overnight 4. 22°C 1 mM IPTG 5 h 5. 22°C 1 mM IPTG overnight 6. 37°C 0.5 mM IPTG 5 h 7. 37°C 0.5 mM IPTG overnight 8. 37°C 1 mM IPTG 5 h 9. 37°C 1 mM IPTG overnight 10. Marker