Team:METU Turkey/Results Discussion/Soluble vs insoluble fraction




BL21 with pKK vector
JM109 with pKK vector
In this experiment, we wanted to understand whether the CooA accumulates in inclusion body. In order to understand that we added urea in our lysis buffer.
We subcultured our cells over night and then passed to scale up. After cultures reached to 0.5-0.6 value at OD600 then the cultures were induced with 1 mM IPTG for 5 hours. After induction, we centrifuged the cultures at 6000g for 15 minutes and lysate our pellets with lysis buffer. Lysis buffer includes 50 mM Tris-HCl- 5 mM EDTA, 100 mM NaCl, 8M Urea, 1.7 mM dithionite, 1 mM DTT, 7.5 uL protease inhibitor. The ph of buffer is important so we adjusted the lysis buffer to pH 7.5. The resuspended cells were lysated with probe sonicator. After the sonication process, the cultures were centrifuged at 20000g for 20 minutes. And, the samples were loaded to SDS-Page. We compared results of lysated cultures with lysis buffer that contains urea and with lysis buffer that does not contain urea.
As seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.