fluorescent proteins expressed in nucleus - (Oct/29/2010 )

Hi,
Does anyone have any experience working with a fluorescent protein (BFP, CFP, etc) that is expressed only in the nucleus?
I'm trying to come up with a few ideas, and I'm thinking histones and lamin, but i'm not sure.

Thanks,
V

vetticus3 on Fri Oct 29 08:59:59 2010 said:

How about adding a nuclear localization signal to GFP? I have used PKKKRKVEDA, worked really well.

rsm

Here's a pic. Not really beautiful, but I hope good enough...

rsm

I'm thinking about using BFP or CFP... but that PKKKRKVEDA sounds interesting. I'll check it out. thanks.

vetticus3 on Fri Oct 29 09:33:51 2010 said:

I wouldn't use CFP or BFP, they are too close to endogeneous protein structures emitting in the same wavelength. CFP maybe for FRET, but then I think there's much better ways than FRET for most applications. If you want to use multiple channels, you can use GFP-YFP-mCherry-tdTomato together (better ask your microscope guys if their confocal can do that...).

rsm

I'm working with gfp zebrafish, and wanted to mark the cells. I was thinking about making the membrane mcherry or ds red and staining the nucleus either blue or cyan. theoretically, a yellow nucleus would work?

vetticus3 on Mon Nov 1 12:44:48 2010 said:

Sure, you can distiguish eGFP and YFP without problems on the newer confocal microscopes (e.g. Zeiss LSM 710). As I said, best ask the guys at your microscope facility (or whoever knows what microscopes you have), if they have the right filters.

rsm