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fluorescent proteins expressed in nucleus


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6 replies to this topic

#1 vetticus3

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Posted 29 October 2010 - 12:59 AM

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

Edited by vetticus3, 29 October 2010 - 01:11 AM.


#2 Rsm

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Posted 29 October 2010 - 01:10 AM

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



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

rsm
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#3 Rsm

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Posted 29 October 2010 - 01:17 AM

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

rsm

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#4 vetticus3

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Posted 29 October 2010 - 01:33 AM

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

#5 Rsm

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Posted 29 October 2010 - 03:05 AM

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


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
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#6 vetticus3

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Posted 01 November 2010 - 04:44 AM

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?

#7 Rsm

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Posted 01 November 2010 - 08:32 AM

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?


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
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