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Hi Vitaly,
With respect to monomers, you and other fans of ECFP should enjoy the
following paper:
Cyan Fluorescent Protein Carries a Constitutive Mutation That
Prevents Its Dimerization. </pubmed/21175224>
Espagne A, Erard M, Madiona K, Derrien V, Jonasson G, Lévy B,
Pasquier H, Melki R, Mérola F.
Biochemistry. 2011 Feb 1;50(4):437-439. Epub 2010 Dec 30.PMID: 21175224
(page 3 mentions the N146I mutation does not prevent YFP
dimerization ... FLIM fans may find of interest PubMed 18975974,
especially Fig 2A-C vs 2D and mTFP's lifetime).
Quantitative FRET references: see other papers from from Richard Day's,
papers from Steve Vogel's lab, and Periasamy's lab. Also:
FRET imaging. </pubmed/14595367>
Jares-Erijman EA, Jovin TM.
Nat Biotechnol. 2003 Nov;21(11):1387-95. Review.PMID: 14595367
Enjoy,
George
On 1/29/2011 9:21 AM, Vitaly Boyko wrote:
> mTFP might be a good donor, however based on my experience, v
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Mike,
>
> mTFP might be a good donor, however based on my experience, venusYFP is not the
> best acceptor for the FRET studies. EYFP with the F64L substitution worked
> better for me. ...though in the very beginning I was in a "blind" love with the
> venusYFP too. Also, the paper did not show any advantage of mTFP over the CeFP.
> In addition, though the CeFP carries A206K substitution, I would recommend to
> add well known monomeric mutations in positions 221 and 223 to CeFP and all
> three to EYFP(F64L).
> As it is difficult to monitor fluorophore concentration on a voxel-by-voxel
> basis, highly accurate, quantitative, comparative FRET measurements study is
> still a challenging task (FLIM inclusive). George, do you have a good reference
> to?
>
> As to the vectors, just do it yourself - it is very simple and quick, PCR is a
> powerful tool and Dave Piston is the best source for the CeFP(A206K).
>
> Good luck,
>
> Vitaly
> 301-515-7833
>
>
>
>
> ________________________________
> From: George McNamara<[log in to unmask]>
> To: [log in to unmask]
> Sent: Fri, January 28, 2011 9:32:26 PM
> Subject: Re: CFP YFP plasmid vectors
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Contact Richard Day, Indiana Univ, ask for the constructs published in:
>
> Characterization of an improved donor fluorescent protein for Forster
> resonance energy transfer microscopy.</pubmed/18601527>
>
> Day RN, Booker CF, Periasamy A.
>
> J Biomed Opt. 2008 May-Jun;13(3):031203.PMID: 18601527
>
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483694/?tool=pubmed
>
>
>
> On 1/28/2011 11:18 AM, Mike Tighe wrote:
>
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>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Would anyone be willing to recommend a source for YFP and CFP plasmid
>> vectors for FRET, preferably compatible with D5Hα. Any feedback would be
>> most appreciated.
>>
>> Thanks!!
>> Mike
>>
>>
>>
>>
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>
>
--
George McNamara, PhD
Analytical Imaging Core Facility
University of Miami
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