After centrifuging my bacteria, I added to the E. coli BL21 DE3 pellet: EDTA 1mM, Urea 6M, lysozyme (1mg / ml), PMSF 1mM, and commercial protease inhibitor tablets 1X diluted in water. At 4 hours and 8 hours I perform thermal shock 15 minutes at -80 ° C and 15 minutes at 4 ° C, then centrifuge and perform western blot of the supernatant.
The bands on my PVDF membrane give the impression that the protein is degraded, can the urea and lysozyme be wrong together? Does anyone have the idea that it could be inclusion bodies that do not allow me to see my protein?
Before centrifuging the bacteria, I grew it in the LB broth with ampicillin and 1% D-glucose, then induced it with IPTG at 0.5mM, 1mM, and 2mM and extracted 5 ml of culture in falcon tubes every hour. , each falcon tube is centrifuged and the pellet is treated with what was said at the beginning.
- Badges
- Science method