I induced a culture (Rosetta gami pLysS) with IPTG ( 0.5 mM and 1 mM) at 37 degrees. Together with it I also kept a control ( uninduced). After lysis, when I run SDS-PAGE gel, I get a band at appropriate position in induced culture pellets, but I also get a faded band at the same position in control as well. Is it possible that my protein gets expressed in control without any induction ?
Hi Deekshi,
Yes, it is possible that you get some leaky expression even under induced condition.
1) You should be sure that it is not the overflow from the test sample into the control sample which is being shown as faded band.
2) Identify your protein by western blot if yo have a tag with your protein otherwise, cut out your band and authenticate with mass spec. The only position of the band does not, for sure, tell you wether you have right protein or not.
regards,
Yes T7 promoter shows leaky expression some times. It is OK until the target protein is not toxic to the host cell. else you have to really take measures to control the leaky expression.
As you are using T7 based expression system, you can control the leaky expression by using 1% glucose (final concentration) in your expression media.
All the best...
Hi there,
Are you actually sure the band observed from non induced culture corresponds to your protein of interest? Have you run the same experiment with cell transformed with an empty plasmid in order to check?
When you induce a culture the final protein expression will be more and inducing a culture is to increase protein production and other hormone production. But uninduced culture also produces the protein but when you quantify you can find that uninduced culture gives less protein compared to induced so only after quantification you can come to a conclusion.
Yes, It is common to get expression in uninduced cells. You need to determine if this is "leaky" expression or the endogenous protein. Using an empty plasmid would be a good control.
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