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 cell imaging. Show all posts
Showing posts with label cell imaging. Show all posts
Wednesday, May 1, 2013
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
Subscribe to:
Posts (Atom)