Hi Kornel Milcarz

I won't recommend fusing multiple tags to a protein as it can hamper its function and cause steric hindrance in the interaction with other proteins.

If the protein is expressed at low levels, maybe you can try an expression vector with a stronger promoter sequence.

If you are visualizing the proteins in fixed cells (like PFA or methanol fixation), then you can amplify the signal of your fused protein using primary and secondary antibodies.

Also, check if the fused protein (NeoGreen) is compatible with the fixative method you used. For example, GFP works best with PFA fixation and not methanol.

I hope this helps.

Best,

Harsh

Thanks for the answer. I want to see my protein during timelapse microscopy (around 24h of recording snapshots) so fixation ain't possibility. Will try to fuse it with HaloTag and maybe with AausFP1 which is quite new FP protein (5x brighter than EGFP, mNG is 3x brigther) so maybe this helps.

Hi! You could try using tandem repeats of split fluorescent proteins, to get brigther signal, but of course then be careful to measure that function is not perturbed. For example see this paper: Article Versatile protein tagging in cells with split fluorescent protein

We used it to tag an endogenously a mammalian membrane protein resulting in brighter signal more suitable for timelapse imaging, and in our case the main function of the protein was not perturbed:

Article Seipin Facilitates Triglyceride Flow to Lipid Droplet and Co...

Veijo T Salo

so if I understand - I need to have my protein fused with GFP10 in native locus (or any other plasmid expressed in my organism) and then plasmid with GFP11xn reperats which expression I can induce by adding somekind of a substrate. In practice, my organism is expressing protein with GFP10 and additionally i can induce expression of GFP11 tandem repeats which can bind to GFP10 and enchance the signal. Do i get it correctly?

Hi!

Almost, but rather your protein of interest should be tagged with tandem repeats of GFP11 and the cell should additionally exrpress GFP10. The size of e.g. 7-GFP11 tag is circa 400 bp, so less than single full GFP (circa 700 bp).

Veijo T Salo

thanks for answer. I might try this approach.