http://blog.allelebiotech.com/2013/04/the-development-of-mneongreen/
This week our most recent publication, “A bright monomeric green
fluorescent protein derived from Branchiostoma lanceolatum” will be
published in Nature Methods. It has already been viewable online for
some time now, here is a link.
We believe this new protein possesses a great deal of potential to
advance the imaging fields through enhanced fluorescent microscopy.
mNeonGreen enables numerous super resolution imaging techniques and
allows for greater clarity and insight into one’s research. As a result
of this we are taking a new approach at Allele for distribution of this
protein, and here we will describe the history of the protein and some
of the factors that led us down this path.
mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology
and the Scintillon Institute through the directed evolution of a
yellow fluorescent protein we offer called LanYFP. LanYFP is a super
bright yellow fluorescent protein derived from the Lancelet fish
species, characterized by its very high quantum yield, however, in its
native state LanYFP is tetrameric. Dr. Shaner was able to monomerize
the protein and enhance a number of beneficial properties such as
photostability and maturation time. The result is a protein that
performs very well in a number of applications, but is also backwards
compatible with and equipment for GFP imaging.
Upon publication there was a question of how distribution should be
structured. How would we make this protein available to researchers in a
simple manner was a very difficult challenge. We also relied heavily
on Dr. Shaner’s knowledge and experience in these matters, as he related
his experiences to us from his time in Roger Tsien’s lab at UCSD. When
the mFruits was published their lab was inundated with requests. The
average waiting period was 3 months to receive a protein and they
required a dedicated research technician to handle this process.
Eventually the mFruits from the Tsien lab were almost exclusively
offered through Clontech. Thus we decided that Allele Biotechnology
would handle the protein distribution and take a commercial approach to
drastically decrease the turnaround time. The next challenge we faced
was how to charge for this protein. Due to the cost of developing this
protein, which was fully funded by Allele, there is a necessity to
recoup our investment and ideally justify further development of
research tools, but we also understand the budget constraints every lab
now faces. From this line of thinking we conceived our group licensing
model; we wanted to limit the charge to $100 per lab. The way this is
fiscally justifiable is having every lab in a department or site license
the protein at this charge, including access to all related plasmids
made by us as well as those generated by other licensed users (Click here for our licensing page).
The benefit we see to this is that the protein is licensed for full
use at a low cost, and collaboration amongst ones colleagues is not only
permissible, it’s encouraged. We saw this as a win-win situation. We
would recoup our cost and invest in further fluorescent protein
research, and our protein costs would not be a barrier to research and
innovation.
The granting of a license to use but not distribute material is not
unique to commercial sources. Although academic material transfer
agreements typically contain specific language forbidding distribution
of received material beyond the recipient laboratory, some researchers
choose to disregard these provisions. Unfortunately through this action
they are disrespecting the intellectual property rights of the original
researchers as well as violating the terms of the legal contract they
signed in order to receive the material. We believe most researchers
choose to respect the great deal of effort that goes into the creation
of research tools for biology and do not distribute any material
received from other labs without their express permission. However for a
company that funds its own basic research our focus is often on the
former example rather than the latter. We believe that this focus
artificially drives up the costs of licensing a fluorescent protein and
obtaining the plasmid, thus we have chosen to believe researchers will
respect our intellectual property as long as we are reasonable in our
distribution which is something we have truly striven for.
Additionally we believe the broad-range usage of a superior, new
generation FP is an opportunity to advocate newer technologies that can
be enabled by mNeonGreen, together with a number of Allele’s other
fluorescent proteins (such as the photoconvertible mClavGR2, and
mMaple). These new imaging technologies are called super resolution
imaging (MRI). They provide researchers with a much finer resolution of
cellular structures, protein molecule localizations, and
protein-protein interaction information. We have started the
construction of a dedicated webpage to provide early adopters with
practical and simple guidance, click here to visit our super resolution imaging portal.
Showing posts with label FP. Show all posts
Showing posts with label FP. Show all posts
Wednesday, May 1, 2013
Friday, July 30, 2010
Allele’s pallet of the super star fluorescent proteins
From AlleleBlogs
http://allelebiotech.com/blogs/2010/07/alleles-pallet-of-the-super-star-fluorescent-proteins/
http://allelebiotech.com/blogs/2010/07/alleles-pallet-of-the-super-star-fluorescent-proteins/
“Photoblog”–just some fun pictures from our notebooks.
- The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.
- Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.
The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.
- The LanFPs express well in bacteria.
Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.
- The FPs fold so strongly that they fluorescence even in SDS-PAGE.
- FPs in SDS PAGE–a closer look
- FPs in gel cassette over UV lights
- FPs in gel cassette under blue LED
The purified FPs can be used as “real time” protein markers.
New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.
Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726
Thursday, June 24, 2010
Brightest Ever Fluorescent Protein
http://allelebiotech.com/blogs/2010/06/brightest-ever-fluorescent-protein-2/
LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:
Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl
LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.
Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.
LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:
Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl
LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.
Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.
Labels:
brightest FP,
fluorescent protein,
Fluorescent proteins,
FP,
FP brightness,
FPs,
Lancelet,
monomer FP,
monomerization,
mTFP1,
mWasabi,
RFP,
YFP
Wednesday, December 16, 2009
mTFP1 is an excellent FRET donor
Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).
In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.
In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.
Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.
(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.
AlleleBlog Admin, by Nathan Shaner
Video of the month (NEW!): Protein Expression Systems on youtube (http://www.youtube.com/watch?v=n81orbUebsQ) and at our protein expression page.
Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)
New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (http://www.allelebiotech.com/shopcart/index.php?c=197&sc=34), 20 infections, only $249 for a limited introduction time.
In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.
In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.
Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.
(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.
AlleleBlog Admin, by Nathan Shaner
Video of the month (NEW!): Protein Expression Systems on youtube (http://www.youtube.com/watch?v=n81orbUebsQ) and at our protein expression page.
Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)
New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (http://www.allelebiotech.com/shopcart/index.php?c=197&sc=34), 20 infections, only $249 for a limited introduction time.
Labels:
cell imaging,
CFP,
FLIM-FRET,
Fluorescent proteins,
FP,
FRET,
FRET acceptor,
FRET donor,
FRET pair,
GFP,
GFP fusion,
mTFP1,
mWasabi,
subcellular localization
Tuesday, June 16, 2009
Allele Will Bring a New Family of Fluorescent Proteins to the Market
Allele has signed an exclusive co-development and marketing agreement with the Swedish high tech company, Innoventus, to work with Dr. Olle Israelsson of the Karolinska Institutet on a novel class of fluorescent proteins.
These proteins were discovered in Amphioxus, a type of small fish that can be found in beach sand, which is believed to be a very primitive cordate species. Compared to jellyfish and coral, from which virtually all of the currently used fluorescent proteins were isolated, Amphoixus are closer to mammalians and their proteins may find great application in human cells and other commonly used animal models. In addition, there are a large number of protein variants that can provide different spectra and other important physical properties such as photostability and photoconvertability.
Allele Biotech’s plan is to first introduce several new fluorescent proteins of different colors to the market as immediate alternatives for fluorescent protein customers. The next step is to continue to evolve and mature these proteins to create more advanced proteins with desired properties suitable for live animal imaging or more advanced applications such as PALM/STORM and SIM. Allele Biotech has on its team of fluorescent protein research staff and collaborators, some of the most highly regarded scientists. With these resources, Allele Biotech plans on committing to long-term development of truly user-friendly fluorescence imaging products.
These new class of fluorescent proteins will be integrated into Allele Biotech’s current products including: retro/lentiviral vectors, baculovirus and bacmam systems, as well as iPSC and RNAi constructs.
These proteins were discovered in Amphioxus, a type of small fish that can be found in beach sand, which is believed to be a very primitive cordate species. Compared to jellyfish and coral, from which virtually all of the currently used fluorescent proteins were isolated, Amphoixus are closer to mammalians and their proteins may find great application in human cells and other commonly used animal models. In addition, there are a large number of protein variants that can provide different spectra and other important physical properties such as photostability and photoconvertability.
Allele Biotech’s plan is to first introduce several new fluorescent proteins of different colors to the market as immediate alternatives for fluorescent protein customers. The next step is to continue to evolve and mature these proteins to create more advanced proteins with desired properties suitable for live animal imaging or more advanced applications such as PALM/STORM and SIM. Allele Biotech has on its team of fluorescent protein research staff and collaborators, some of the most highly regarded scientists. With these resources, Allele Biotech plans on committing to long-term development of truly user-friendly fluorescence imaging products.
These new class of fluorescent proteins will be integrated into Allele Biotech’s current products including: retro/lentiviral vectors, baculovirus and bacmam systems, as well as iPSC and RNAi constructs.
Labels:
Amphoixus,
fluorescence imaging,
fluorescent protein,
FP,
FPALM,
GFP,
HHMI,
Janellia Farm,
mTFP1,
mWasabi,
new FP,
novel FP,
PALM,
SIM,
STORM
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