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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:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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.
>
>
>
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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/
>
>
> 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
>> *****
>>
>> 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:[log in to unmask]] On Behalf Of George McNamara
>> Sent: 14 September 2012 01:57
>> To: [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
>> *****
>>
>> Hi Simon,
>>
>> likely riboflavin and possibly other flavins. See
>> 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>
>>
>>
>> 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:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> 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
>>> Has anyone tried this? Does anyone know how it works?
>>>
>>> Thanks,
>>> Simon
>>>
>>>
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