2 replies to this topic

[dancedive]

Hello,

I hope this is the correct place to create the topic. I have a question about tagging a protein with GFP (fusion protein) to track its localization in the cells. Here it goes:

In almost every article researchers say, "we tagged protein x with GFP by using transiently transfected vector y" but they seldom explain the effect of expression levels of engineered protein and its biological effects.

If, let's say, I want to tag a protein whose intracellular level affects organelle morphology (for instance Mfn2 increases mitochondrial fusion) how can I achieve tagging the protein without flooding the biological system with excess amount of protein of interest (e.g. Mfn2). In other words, how can I tag protein of interest with GFP without overexpressing it?

transient transfection vectors are all containing strong promoters for high level expression of proteins of interest. This is good for studies involving protein isolation, but how can I tag a protein with GFP in the cells by transient transfection?

I hope it was clear enough to express my question.
Thanks for the answers in advance

Edited by dancedive, 03 February 2012 - 10:45 PM.

[scolix]

To get a low level expression, you will have to use weak promoters in recombinant viruses. Or make stable cells, select cells as you wish.

You can also try tet on or off system, to regulate expression.

[bob1]

Be aware also that tags can interfere with the function of the tagged protein - this is especially the case if the tag is large and in close proximity to the protein of interest.