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Problems with Bacterial Induction: NO Induction


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#1 plasmodel

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Posted 17 January 2011 - 03:42 AM

So, I am trying to induce a protein in E. coli. So far, I have tried growing ON cultures at 27, 28 and 37 C. 27 and 28 were o/n and 37 for 3 hours. I have been doing small scale (4 ml approx) before I move to bigger ones. So, I grow a starter culture on at 37 and then, bring it upto OD 600 of around .8 and then, induce them. I have also played around with IPTG concentrations (0.5 mM and 1.0 mM). My positive control has induction, when I look at it using anti-his antibodies but my protein doesn't. But, when I use antibodies against my protein, I see faint bands (but not increasing as should be seen if induced).
at it using anti-his antibodies but my protein doesn't. But, when I use antibodies against my protein, I see faint bands (but not increasing as should be seen if induced).
I have been using Bl21 (DE3) but I am going to try with plysS tomorrow. Can anyone help?

Also, I had two questions: (1). Why does one play around with IPTG concentrations; shouldn't it get saturated at some point and adding more is not going to help?
(2). How do you separate insoluble from soluble fraction? I have been lysing, sonicating and centrifuging it for like 5 minutes at max speed. But, the bands for my postive control protein show up in both fractions.

#2 protolder

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Posted 17 January 2011 - 11:13 PM

So, I am trying to induce a protein in E. coli. So far, I have tried growing ON cultures at 27, 28 and 37 C. 27 and 28 were o/n and 37 for 3 hours. I have been doing small scale (4 ml approx) before I move to bigger ones. So, I grow a starter culture on at 37 and then, bring it upto OD 600 of around .8 and then, induce them. I have also played around with IPTG concentrations (0.5 mM and 1.0 mM). My positive control has induction, when I look at it using anti-his antibodies but my protein doesn't. But, when I use antibodies against my protein, I see faint bands (but not increasing as should be seen if induced).
at it using anti-his antibodies but my protein doesn't. But, when I use antibodies against my protein, I see faint bands (but not increasing as should be seen if induced).
I have been using Bl21 (DE3) but I am going to try with plysS tomorrow. Can anyone help?

Also, I had two questions: (1). Why does one play around with IPTG concentrations; shouldn't it get saturated at some point and adding more is not going to help?
(2). How do you separate insoluble from soluble fraction? I have been lysing, sonicating and centrifuging it for like 5 minutes at max speed. But, the bands for my postive control protein show up in both fractions.

Hola,Your expression is quasi constitutive because you havenīt any brake to it if you are using LB, TB or any media without glucose,so the cAMP levels are high. Probably if you add glucose 0.5 g/l (cAMP low)you can control the difference before and after induction. If you change to pLys and donīt control it you have to use BL21 lac I wich strongly repress expression untill induction. One inducer molecula binds to one site of promoter, so to a good induction you have to add enouhg molecules as promoters has. If you add low amount, only few plasmids inside the bacteria will induce and little expression you will see, if you add enough or an excess of inducer, all the plasmids were activated and you have the maximum of expression and the excess of inducer will be free withouth bound any promoter. And the end, the sonication is fine, but wash the pellet once tryting to see more clair difference between both soluble and insoluble fraction. Buena suerte




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