what is the logic behind using an uninduced sample as a negative control in protein expression experiments? why cant we use neat vector???? I'm getting bands at the expected size on SDS PAGE of my protein and there is considerable difference between the level of expression between induced and uninduced sample but my supervisor asked me to run neat vector samples both induced and uninduced along with my cloned gene of interest and use it as a negative control for the experiment. I got very unexpected results, my neat vector also shows the band of the same molecular weight as my protein of interest( i have checked my cloned gene and expression vector by digestion, they show correct bands of expected size). Could this protein band on SDS PAGE of unknown bacterial protein?? My supervisor says that you should not get any band same size of your protein. I have always seen in papers uninduced sample of cloned gene of interest is always used as a negative control????
Hi Anam,
your supervisor is spot on. A neat vector control is basically the best negative control for protein expression experiments. You have to consider that no matter which transfection method you use it will always ends up influencing cell behavior to some degree (as seen in your experiment were suddenly you see a band at the exact same height as your protein of interest). So, strictly speaking you can not really compare transfected cells to untreated ones. I do not know which protein you are trying to detect but if it is a protein your cells can express endogenously it is possible that the transfection itself stimulates its production? How do you detect your protein of interest? Are you using a specific antibody or is your detection more unspecific like only a coomassie/silver stain? If the problem is endogenous protein expression you should add a tag to your protein of interest. That way you can clearly differentiate between endogenous and transfected proteins.
Other than that I suggest to make sure that your neat vector stock solution is not contaminated with trace amounts of your expression vector. You can check this by performing a PCR and trying to amplify a region of the cDNA of the protein.
As for why you do not see neat vector controls in papers more often: Well, I guess it depends on the expressed protein. For example, if you transfect insect cells to express a protein that only exists in humans and detect it with a specific antibody in Western Blot I think that using untreated cells as a control is acceptable. The same goes for proteins coupled to a specific tag. It is not as perfectly scientifically accurate as using a neat vector control but most people will accept it. However, with proteins that are produced endogenously or proteins you can only verify in unspecific ways you have to be more careful since you have a bigger chance of detecting artifacts.
Todor Tschongov I'm trying to express HBV surface gene ligated with prset B vector in prokaryotic expression system. The vector has his tag at its N terminal. I have used coomasie brilliant blue staining method for detection
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