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see this article from Lin (& Tsien) in Nature Methods:
http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2171.html
Clover photostability seems to be quite worse than GFP though.
Christophe
On Fri, Sep 14, 2012 at 8:58 PM, Kurt Thorn <[log in to unmask]> wrote:
> *****
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>
> What is Clover GFP? I can't find much information about it on Google.
>
> Thanks,
> Kurt
>
>
> On 9/14/2012 3:49 AM, George McNamara wrote:
>
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>>
>> Hi Simon,
>>
>> Your cells might not need the 100x excess of riboflavin present in
>> "standard" DMEM, your background could be reduced. The Essen tech note I
>> mentioned lists:
>> DMEM 0.4 mg/L riboflavin ... 43.6 units fluorescence
>> Eagles MEM 0.1 mg/mL ... 12.9
>> F12K 0.04 ... 5.4
>> EBM 0.004 ... 3.7
>> Riboflavin (alone) 0.4 ... 58.7 (perhaps suggesting that culture
>> media quenches riboflavin or it gets converted in part to something less
>> fluorescent?)
>> Contact Essen if you want the entire tech note.
>>
>>
>> If you absolutely require a green fluorescent protein, spend the time to
>> switch to the new Clover or "V6" from Steven Vogel (available from
>> addgene.org as VVVVVV).
>> If you do not need green, switch to tdTomato or the new mRuby2.
>>
>>
>>
>> Choose Destination
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>> exquery/spam_note.html>
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>> Nat Methods. <#> 2012 Sep 9. doi: 10.1038/nmeth.2171. [Epub ahead of
>> print]
>>
>>
>> Improving FRET dynamic range with bright green and red fluorescent
>> proteins.
>>
>> Lam AJ </pubmed?term=Lam%20AJ%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> St-Pierre F </pubmed?term=St-Pierre%20F%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Gong Y </pubmed?term=Gong%20Y%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Marshall JD </pubmed?term=Marshall%20JD%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Cranfill PJ </pubmed?term=Cranfill%20PJ%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Baird MA </pubmed?term=Baird%20MA%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> McKeown MR </pubmed?term=McKeown%20MR%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Wiedenmann J </pubmed?term=Wiedenmann%20J%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Davidson MW </pubmed?term=Davidson%20MW%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Schnitzer MJ </pubmed?term=Schnitzer%20MJ%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Tsien RY </pubmed?term=Tsien%20RY%**5BAuthor%5D&cauthor=true&**cauthor_uid=22961245>,
>> Lin MZ </pubmed?term=Lin%20MZ%**5BAuthor%5D&cauthor=true&**
>> cauthor_uid=22961245>.
>>
>>
>> Source
>>
>> 1] Department of Bioengineering, Stanford University, Stanford,
>> California, USA. [2] Department of Pediatrics, Stanford University,
>> Stanford, California, USA.
>>
>>
>> Abstract
>>
>> A variety of genetically encoded reporters use changes in fluorescence
>> resonance energy transfer (FRET) to report on biochemical processes in
>> living cells. The standard genetically encoded FRET pair consists of CFPs
>> and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range,
>> phototoxicity from the CFP excitation light and complex photokinetic events
>> such as reversible photobleaching and photoconversion. We engineered two
>> fluorescent proteins,* Clover and mRuby2*, which are the brightest green
>> and red fluorescent proteins to date and have the highest Förster radius of
>> any ratiometric FRET pair yet described. Replacement of CFP and YFP with
>> these two proteins in reporters of kinase activity, small GTPase activity
>> and transmembrane voltage significantly improves photostability, FRET
>> dynamic range and emission ratio changes. These improvements enhance
>> detection of transient biochemical events such as neuronal action-potential
>> firing and RhoA activation in growth cones.
>>
>> PMID:
>> 22961245
>>
>>
>> PLoS One. <#> 2012;7(5):e38209. Epub 2012 May 30.
>>
>>
>> Fluorescence polarization and fluctuation analysis monitors subunit
>> proximity, stoichiometry, and protein complex hydrodynamics.
>>
>> Nguyen TA </pubmed?term=Nguyen%20TA%**5BAuthor%5D&cauthor=true&**cauthor_uid=22666486>,
>> Sarkar P </pubmed?term=Sarkar%20P%**5BAuthor%5D&cauthor=true&**cauthor_uid=22666486>,
>> Veetil JV </pubmed?term=Veetil%20JV%**5BAuthor%5D&cauthor=true&**cauthor_uid=22666486>,
>> Koushik SV </pubmed?term=Koushik%20SV%**5BAuthor%5D&cauthor=true&**cauthor_uid=22666486>,
>> Vogel SS </pubmed?term=Vogel%20SS%**5BAuthor%5D&cauthor=true&**
>> cauthor_uid=22666486>.
>>
>>
>> Source
>>
>> Section on Cellular Biophotonics, Laboratory of Molecular Physiology,
>> National Institute on Alcohol Abuse and Alcoholism, National Institutes of
>> Health, Rockville, Maryland, United States of America.
>>
>>
>> Abstract
>>
>> Förster resonance energy transfer (FRET) microscopy is frequently used to
>> study protein interactions and conformational changes in living cells. The
>> utility of FRET is limited by false positive and negative signals. To
>> overcome these limitations we have developed Fluorescence Polarization and
>> Fluctuation Analysis (FPFA), a hybrid single-molecule based method
>> combining time-resolved fluorescence anisotropy (homo-FRET) and
>> fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm
>> proximity gauge), brightness (a measure of the number of fluorescent
>> subunits in a complex), and correlation time (an attribute sensitive to the
>> mass and shape of a protein complex) can be simultaneously measured. These
>> measurements together rigorously constrain the interpretation of FRET
>> signals. Venus based control-constructs were used to validate FPFA. The
>> utility of FPFA was demonstrated by measuring in living cells the number of
>> subunits in the ?-isoform of Venus-tagged calcium-calmodulin dependent
>> protein kinase-II (CaMKII?) holoenzyme. Brightness analysis revealed that
>> the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from
>> 10-14 in individual cells. Homo-FRET analysis simultaneously detected that
>> catalytic domains were arranged as dimers in the dodecameric holoenzyme,
>> and this paired organization was confirmed by quantitative hetero-FRET
>> analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ±
>> 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the
>> reduction in subunit number, catalytic domains were still arranged as pairs
>> in homogenates. Thus, FPFA suggests that while the absolute number of
>> subunits in an auto-inhibited holoenzyme might vary from cell to cell, the
>> organization of catalytic domains into pairs is preserved.
>>
>> PMID:
>> 22666486
>>
>>
>> I am a bit disappointed Vogel's group did not go for V8 (a well known
>> drink) or V12 - the latter either as a polypeptide or with inducible
>> dimerization domain. V12 since the goal of this paper is to quantify the
>> number of subunits in CaMKIIalpha, which turns out to be 12 (+/- a few) as
>> described in http://www.ncbi.nlm.nih.gov/**pmc/articles/PMC3364239/**
>> figure/pone-0038209-g004/<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364239/figure/pone-0038209-g004/>
>>
>> On 9/14/2012 4:32 AM, simon walker wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>> Thanks for the various responses. Yes, I'd seen the Bogdanov paper and
>>> the Evrogen medium and thought that might be worth a try. The problem we
>>> have is that for our assay the culture medium is absolutely critical (it's
>>> not just a case of keeping cells alive), so we can't use a minimal
>>> HEPES-based buffer. I am interested to know what is in the 'BackDrop'
>>> solution. We can't use it unless we're fairly confident it's not going to
>>> affect our assay.
>>> Simon
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**
>>> LISTS.UMN.EDU <[log in to unmask]>] On Behalf Of George
>>> McNamara
>>> Sent: 14 September 2012 01:57
>>> To: [log in to unmask]**EDU<[log in to unmask]>
>>> Subject: Re: Background fluorescence problem
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>> *****
>>>
>>> Hi Simon,
>>>
>>> likely riboflavin and possibly other flavins. See
>>> http://www.evrogen.com/**products/medium_DMEM_gfp/**
>>> medium_DMEM_gfp.shtml<http://www.evrogen.com/products/medium_DMEM_gfp/medium_DMEM_gfp.shtml>
>>> and the Bogdanov et al paper referenced at the bottom of the page;
>>>
>>> * Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov
>>> S, Lukyanov KA. Cell culture medium affects GFP photostability: a
>>> solution. Nat Methods. 2009; 6 (12):859-60. / pmid: 19935837
>>> <http://www.ncbi.nlm.nih.gov/**entrez/query.fcgi?cmd=**
>>> Retrieve&db=PubMed&list_uids=**19935837&dopt=Abstract<http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19935837&dopt=Abstract>
>>> >
>>>
>>>
>>> Their solution: incubate cells in miedia without (or with low, if
>>> needed) riboflavin for a day.
>>>
>>> As a bonus, riboflavin quenches (FRET?) and/or transiently photoconverts
>>> GFP to red fluorescence (might be mostly dark states):
>>>
>>> Condensed mitotic chromosome structure at nanometer resolution using
>>> PALM and EGFP- histones.</pubmed/20856676>* Matsuda* A, Shao L, Boulanger
>>> J, Kervrann C, Carlton PM, Kner P, Agard D, *Sedat* JW. PLoS One. 2010 Sep
>>> 15;5(9):e12768. PMID: 20856676
>>>
>>>
>>> If you contact Essen Biosciences, they will (hopefully) give you a copy
>>> of their application note on the concentrations of riboflavin in many
>>> culture media and correlation with fluorescence of those media. Speaking of
>>> Essen - they finally introduced a dual green+red fluorescence Incucyte.
>>>
>>> Enjoy,
>>>
>>> George
>>>
>>>
>>>
>>> On 9/13/2012 11:04 AM, Simon Walker wrote:
>>>
>>>> *****
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>>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>>>> *****
>>>>
>>>> Dear List,
>>>> We are imaging very weakly fluorescent live cells (expressing GFP) on
>>>> a wide- field system and having issues with a source of background
>>>> fluorescence.
>>>> When we look at our cells under epi-illumination we see a rapid drop
>>>> in a weak background signal (not where the cells are) that fully
>>>> recovers over a ~10 s period after the illumination light is switched
>>>> off. Our experiments require the use of DMEM as the imaging medium
>>>> and this is the likely cause of problem. It appears that something in
>>>> the medium is sticking to the coverglass. It's not phenol red as the
>>>> effect is seen with both phenol red-containing and phenol- red-free
>>>> DMEM. Does anyone know what else it could be? Has anyone else seen
>>>> anything similar? We're wondering if it could be riboflavin which is
>>>> in the DMEM we're using. Would this stick to glass?
>>>>
>>>> I've seen that Life Technologies now market a substance that allegedly
>>>> surpresses background fluorescence in DMEM:
>>>> http://products.invitrogen.**com/ivgn/product/R37603<http://products.invitrogen.com/ivgn/product/R37603>
>>>> Has anyone tried this? Does anyone know how it works?
>>>>
>>>> Thanks,
>>>> Simon
>>>>
>>>>
>>>> The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT
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