在第二阶段的实验水平较低的细胞治疗丝裂霉素C和伴随免疫印迹支出抗体培育对大肠杆菌素Ia兔子。巷1标记,巷2纯化蛋白,巷3 1 h感应,巷4 2 h,lane5 3 h,巷5 5 h。
In the second phase of the experiment the cells were treated with lower levels of mitomycin C and an accompanying western blot spending antibodies nurtured in rabbits against colicin Ia. Lane 1 markers, lane 2 purified protein, lane 3 1h induction, lane 4 2h, lane5 3h, lane 5 5h.
The colicin X gene was amplified through the use of primers by PCR for creating two other genes known as Ncol and Xhol. The fragment of PCR had to go through cloning with a pET28avector leading towards providing the C-terminus with a His tag illustrating no inhibition of pore forming toxins activity (Braun et al 1994). Further, on a chromatography system, this protein was run by following the steps of protocol. In column loading buffer with 40 ml mixture inclusive of sodium phosphate, sodium chloride and Imidazole (50 MM, 300 MM and 10 MM respectively), the pellet was suspended again. To this mixture inhibitors of protease with DNase and RNase were added before sonication. The mixture of all these elements were added again and the incubation of ice extraction over a period of 45 minutes for ensuring contamination of DNA and RNA were eliminated. Those pelleted cells through the process of centrifugation within JA20 at 4 degree Celsius were uses to which a filter was added and the inhibitors of protease were added.