I constructed 2 expression plasmid as follows:
1, designed forward primer: TT+EcoRV+GC+EcoRI+GG+(1-14)F
reverse primer: TT+XhoI+(1062-1043)R
EcoRV and XhoI are designed for pCMV-Tag2A
EcoRI and XhoI are designed for pCMV-Myc
2, amplified this PCR product from another expression vector with no tag.
3, TOPO cloned it...finally got the plamid with this insert.
4, digestion, purification and ligation to the new vectors
5, finally got the right plasmids and sequenced them.
it is ok with all above, but when I used these two tagged plasmid in Reporter assay or western blot. I found both didnt work in the reporter assay (compared with the one without tag) and only Myc-tagged one showed clear band in western, Flag-tagged one showed very very weak band.
I dont know: 1, why Flag tagged one didnt work? 2, why Myc tagged one worked in western but not in Luc assay?
3, does tag affect protein function? I am also using many other expression plasmids with or without tags, they all work well.
Thanks in advance.
Contructed expression plasmid but didnot work
No replies to this topic