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three- color analyses - (Sep/05/2007 )

Hi,
I 'm trying to do a three- color analyses. I'm planning to use EGFP and DsRed. Does anyone know which fluorescence protetin I can use in conjunction with EGFP and DsRed?

Thanks,
Joe

-veryslowjoe-

QUOTE (veryslowjoe @ Sep 6 2007, 07:59 AM)
Hi,
I 'm trying to do a three- color analyses. I'm planning to use EGFP and DsRed. Does anyone know which fluorescence protetin I can use in conjunction with EGFP and DsRed?

Thanks,
Joe


This paper may help you a little. What they discussed in the paper is fluorescent proteins. So if you want dyes, that is a different story. I believe somewhere in the forum, there are people talking about various types of dyes. For FarRed there are Cy5, AlexaFluor647. In any case, the DsRed is a bit close to the FarRed range, when we check the spectrum, there is some crossover. So be careful with it and better to take sequential. You can use the UV range, too. Make sure that you have the proper filter and check the spectrum, excitation/emission so that there is no bleed through.

-Almasy-

QUOTE (veryslowjoe @ Sep 5 2007, 04:59 PM)
Hi,
I 'm trying to do a three- color analyses. I'm planning to use EGFP and DsRed. Does anyone know which fluorescence protetin I can use in conjunction with EGFP and DsRed?

Thanks,
Joe


you can use UV (<400 nm; often used for Hoe or DAPI nuclear stain), CFP, YFP or far red (633 nm) additionally to 488 and 546 nm; of course, you should do sequential, non-parallel imaging

-The Bearer-

QUOTE (The Bearer @ Sep 6 2007, 06:58 PM)
QUOTE (veryslowjoe @ Sep 5 2007, 04:59 PM)
Hi,
I 'm trying to do a three- color analyses. I'm planning to use EGFP and DsRed. Does anyone know which fluorescence protetin I can use in conjunction with EGFP and DsRed?

Thanks,
Joe


you can use UV (<400 nm; often used for Hoe or DAPI nuclear stain), CFP, YFP or far red (633 nm) additionally to 488 and 546 nm; of course, you should do sequential, non-parallel imaging


I am not sure about the YFP. The emission peak is quite close to GFP (527 for YFP and 507 for GFP), the majority of their spectrum overlap, at least 70-80% . Unless you have very specific filter AND doing sequential, you run high risk of bleed through. We have tried with these two, really no good.
Also, as you can see in the paper, the DsRed is also not recommended. They dim too fast. I think you can find that others in the forum have complained about it before. Cherry or Tomato is way better, or maybe Strawberry (this I haven't tried). As for the CFP, I remember I have read somewhere that it need a bit special in either fixing or permeablizing or mounting, but I am unable to find the information right now. If anyone knows anything about it, please let us know? Thank you very much in advance.

-Almasy-