Western Blot can not detect the expression of protein, although the green fluorescence is expressed. Why ?
The vector is CMV promoter and the sequence is that eGFP-2A site-Myc-insert.
There are some MCS between 2A Site and Myc-tag.The cell line is 293T.The insert gene is a kind of polymerase which is from avian influenza.
I can perceive the green fluorescence frome the 293T cell by fluorescence inverse microscope.However,i can not detect the expression of my protein by Western Blot using Myc-antibody.Is the reason that the protein which is a kind of virus's polymerase is a toxic protein for 293T cell and the protein is degraded?
If the myc tag falls into frame and there is no stopcodon in between the MCS shouldn´t matter. Did you try to use a GFP antibody? Also if you have an imaging system with which you can detect fluorescence you can check wheather the protein is at all present via GFP fluorescence. That way you can narrow it down more, so far I would try to check separately at which point things go wrong
1. Is the myc-tag expressed as part of the fusion protein you can observe at some point, I don´t know the situation, but if you just used someones plasmid or a stable transfected cell line you got from somewhere, I would sequence the for myself.
2. If so, is the sample you use for your Western Blot fluorescent (if you have acess to a nanodrop you should not have to sacrifice a lot of sample for that)?
3. If the protein with myc-tag is in the sample, is it just a problem with the particular antibody, so does another antibody (like anti-GFP) work?
If you can figure out, where you lose the myc-tagged protein, it is much easier to find the cause. Right now there are so too many possibilities.
Is the antibody used for WB the good one?
- Monoclonal or polyclonal, since you are using a protein fusion I would suggest a polyclonal.
- Which part of the protein sequence is your antibody targeting? ( this is written in the technical sheet and should be easily accessible). I would suggest to target part of the prot sequence away from the fusion site.
-As mentioned earlier an anti-GFP would be the best bet.
Best,
Romuald
Hi Zhang,
Are you sure the WB-Ab is able to detect the protein on the blot? Have you tested it for this application?
I have done RT-qPCR and the GFP's mRNA transcription level is the same as interest gene.Other protein which is located at the same situation in plasmid and tagged myc can be detected by Western Blot.
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