Hi, recently I tried to express my genes (from plant) into Rosetta De3 pLysS using this protocol:
1. 1% overnight culture into a new media + antibiotic, incubated to grow until OD600= 0.5
2. Induce at 19°C, overnight with 0.1mM, 0.3mM and 0.5mM IPTG.
3. On the next day, 1ml culture was taken and spin to separate pellet and supernatant. Supernatant is discarded as protein is intracellular (discovered before during expression using different competent cells)
4. Pellet was resuspend in 1ml of lysis buffer (KH2P04, K2HPO4, NACl, KCl, glycerol, Triton X-100 and H2O, pH 7.8) by vortex.
5. Resuspend pellet was sonicated for 5 min (30s on, 30s off) then spin to separate soluble protein (in supernatant) an insoluble protein (in pellet)
5. Pellet was resuspend with 8M urea
6. SDS PAGE analysis was conducted upon addition of sample buffers
From the gel, there is no prominent band at the appropriate size for my genes at all. Why does this happen?
Thank you in advance for answering
Hi Fatin,
From my understanding, you are trying to express a plant protein in bacteria. kindly check if it has any PTM first. There could be many reasons why you did not observe any expressed protein. Please share the SDS-PAGE gel picture with appropriate labels and protein size as the information provided is insufficient. According to the induction conditions you have mentioned, have you tried it in other temperatures too (37 and 25) if so attach the gel pictures and also run different pellet and supernatant fractions, and do not discard it before you confirm it as intracellular.