2 replies to this topic

[pinpig]

I am going to constuct a stable cell line that overexpress one gene. I would like to ask whether I have to to choose a plasmid with the tags. The tags are more helpful for further studies? any harm? Thank you very much!

[bob1]

It really depends on what you are trying to look at.  Any tags, especially such as GFP which is huge, may interfere with nuclear/mitochondrial/any structural transport, or cause steric hindrance for interactions, block antibody binding etc.  Overproduction of some proteins may affect what happens to them, or cause the cells to die etc.

[laurequillo]

As Bob says it depends on the tag, if it is in N or in C terminus and on your protein. The best you can do is first to check it in transient transfection, and see ifi ts behaviour in WB and in IF is the expected one (you can compare differents tags). If not, I would try with flag in N and C terminal