I have bacteria transfected with cloned pGEX-6-P-1 vector, I need to induce expression using IPTG at low temperature to minimize formation of inclusion bodies and increase solubility of my protein , so which is better to start inducing at the Bacterial Log phase or at the stationary phase?
what the best protocol for induction of a batch volume around 500 ml L.B?
Hi there,
There is no general rule to follow as the result of the experiment depends on the nature of the target... But it is admitted that growing cell is producing more than non growing cell. So midlog phase (OD 0.8 to 1) is a good starting point. As induction will occur at low temperature it is important to have biomass prior induction as the cells won't grow a lot during induction.
If you use 500mL culture, inoculate it with an overnight culture so as OD is 0.1 (1/100 to 1/50 of the stationary phase culture) then grow cells at 37°C until OD 0.8 then induce expression using an IPTG concentration which may vary (start with 0.5mM) and place the culture overnight at the desired expression temperature which also may vary (we routinely do 15°C for 16h in our lab)...
I agree with the IPTG concentration recommended buy dominique, but in my experience, inducing the cells at a lower OD value is preferable. In our lab, we prefer an OD of around 0.6.
Just a few remark to add: 0.5mM IPTG is not a low concentration. When testing this parameter, one can decrease it down to 0.05mM. Concerning OD for induction, the optimal value will vary from one spectrophotometer to one other as what is actually measured is light diffusion rather than absorption and diffusion measure depends on the apparatus geometry which does vary a lot from one to an other (diffusion measure will increase with the distance between sample and detector).
Dear All:
please find the attached file
Thanks for your advice, actually I succeeded in inducing my protein expression at 18c for 15 h using IPTG 0.07 % final concentration,
But , I am facing a great problem now as my purified protein (expected to be at 42KDa) contain one strong non specific band around (60KDa), When this purified protein product had been checked by Western Blot tech. using HRP ani GST , only one band appeared corresponding to my band while the other none specific did not appear confirming that it is not GST bound one.
So I suggested that my washing process of the Beads was not optimized.
After repeating the purification process , I increased the bounded beads washing process to be 5 times with cold 1x pBS before elution, unfortunately I obtained the same result again , So I can not immunize my animal with this protein
what is the solution of this problem?
please find the attached file
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