cloning of non-coding region for protein expression - protein expression (Jan/08/2010 )
I am trying to see the function of my non-coding region in protein expression. I have clone my gene and its non-coding region upstream of 5'UTR to find if non-coding region plays any role in expression of cloned protein. I have other clones with no non-coding region just 5'UTR and coding region. Has anybody cloned these type construct in mammalian expression vector and does it work? I am using pcDNA3.1 from invitrogen. The positive control is beta-galactosidase plasmid, which is co-transfected with my plasmid in HEK293. These both the plasmid has c-myc tag. When i do antibody staining for c-myc on westernblot it shows band crosspoding to bet gal but not my protein. Does anybody know if non-coding region interfere with protein expression or any one explain why i don't see my protein expression or any suggestions?
I have seen many times people make vectors with 5' UTR to have better transcription and eventually better protein expression.