Hello Everyone,
Recently I engineered a new fusion gene which contained a promoter region fused to a protein which is a hybrid of two proteins (genetically engineered in a certain fashion). It is expressed in bacteria and is a membrane protein.
Before I test the functionality of this engineered protein (for what it was designed to do), I would like to confirm that the protein is being produced normally. In other words, if my functional assays do not work out, at least I know the protein is being produced properly, and the gene of interest is actually present.
There is not easy functional assay to asses this since it is a new protein. I was thinking of perhaps doing a RT-PCR for the fusion gene of interest to see whether it is being produced. Is there any other control tests that should be done to confirm this protein? Would some type of protein extraction and SDS-PAGE be required?
Thanks.
Adam
A Western blot would show whether the protein is being produced. For this, you require an antiserum or antibody recognizing some epitope in the protein. You also need a negative control sample from cells that do not express the protein, such as cells that have the empty vector. If there is no available antiserum or antibody, then you can have one created by a specialist company, or make one yourself, using a synthetic peptide based on the protein sequence as the antigen.
- To confirm production of your fusion protein I have always peerformed small scale production with conditions on trial and run ~ 1ug on an acrylamide gel which I have then coomassie stained and de stained with methanol acetic acid (3:1)
- That should be suuficient for a qualitative answer, i.e. is it there or not
- Remember though to also load un induced controls so the band that increases of a specific size can be deduced to be your induced (fusion) protein
The RT-PCR will confirm that the fusion gene is there. But it will not tell you if the new gene is functioning.
For that you have to make a Western blot on the new protein.
1. I actually don't think western blotting is the best option
2. It will tell you if the gene has been expressed but will not show you how much it has been enriched by induction
3. For that the simplest and most effective way as said is to examine the protein on an acrylamide gel by comassie staining
4. At that point you can unambiguously confirm identity by Western blotting as said but western blotting is only effective in concert with comassie staining which will more effectively demonstrate efficiency of induction
I agree with Laurence Stuart Dawkins-Hall that Coomassie staining should be tried to demonstrate expression of the protein, if it it expressed at a high enough level to detect that way. Use a membrane preparation rather than a total cell extract, because that enriches for membrane protein. That should also remove the selection marker protein, which is also expressed from the plasmid but is normally a soluble protein. Consider also the possibility that the protein may not get incorporated into the membrane, but may end up as inclusion bodies. Do an inclusion body prep and look for it there. Use a Western blot, if possible, to confirm that the identified protein is the right one and to see if there are truncated or degraded forms of the protein also present.
Thank you everyone for your input. I think to start I will do a standard SDS-PAGE with a membrane protein extraction protocol. By growing it in a certain metal, I can increase the expression of the protein, and hence, this can be a way for enriching that protein.
I could use an OmpC antibody to further confirm..... though I must realize that there will be multiple of them since OmpC is also a protein naturally produced, and part of the domain is in the fusion protein.
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