I made a recombinant plasmid and tested it by colony PCR and double restriction digest, both of them showed the construct was correct but the western blot always cannot show the protein I wanted was expressed ( The results I got were either no bands or only GFP tag was expressed). Does anyone meet the same situation? I'm doubting if it is due to WB transfer failure (but I can see the marker bands) Does that make sense?
there are so many possibilities for your protein not to be expressed that it is especially necessary first to check the sequence of your plasmid construction then try to get a positive control for your western blot (e. coli expressed protein for example) then check the transfection efficiency then....
What kind of a fusion protein did you make? protein-GFP or GFP-protein? Double check to make sure there isn't a stop codon in there and that the reading frame is correct for the full fusion protein.
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