You can help EcoliWiki by editing the content of this page. For information about becoming a registered user and obtaining editing privileges, see Help:Accounts.
Protocol adapted from Molloy et al. (2000) 
- 50mL culture at A550 of 0.2. Induce expression for 30 mins. Harvest.
- Resuspend pellet in 1mL FPB (100mM Na2HPO4, 5mM EDTA, 100mM KCl; pH 8.0) and add 2μL 0.5M DTT, 5μL protease inhibitor (commercially available), 10μL 0.1M PMSF (optional), 20μL 1M MgCl2, 5μL 10mg/mL DNase.
- Pass sample through the French press. Centrifuge at 14 000 rpm for 2 mins to remove cell debris and unlysed cells.
- Divide the supernatant into 2 samples & centrifuge in a TLA 100.3 rotor for 1 hr at 4oC at 50 000 rpm.
- To one of the pellets, add 500μL 1% Triton X-100. To the other pellet, add 500μL 0.1M Na2CO3 (MADE FRESH!).
- Put a tiny stir bar in each and mix until the pellet is completely dissolved (about 3h).
- Centrifuge again in TLA 100.3 at 4oC for 1 h at 50 000 rpm.
- Remove supernatant and resuspend pellets in 500μL sample loading buffer and boil.