Cytoplasmic Extract

1. Start with 3 - 250 mL cultures.
2. Pellet cells @4000 RPM for 4 min.
3. Resuspend in 10 mL of autolysin and shake in the light until they can be seen using NP-40 that they have lost their walls. (Typically 1 - 1 ½ h).
4. Aliquot into 15 mL conical tubes and spin @4000 RPM for 7 min.
5. Resuspend pellet in one volume of NaLow.
6. Add NP-40 to a final concentration of 0.5%.
7. Optional Vortex dependent on cell wall loss.
8. Aliquot into microfuge tubes and spin @14,000 RPM for 15 min.
9. Collect supernatant into ultracentrifuge microtubes; balance; spin @70,000 RPM for 1 h.
10. Dialyze the supernatant for 1 ½ h with two changes of NaLow.
11. Spin @14,000 RPM for 15 min.
12. Do protein assay and use as needed.