Showing posts with label FP. Show all posts
Showing posts with label FP. Show all posts

Wednesday, May 1, 2013

The Development of mNeonGreen

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.

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/


“Photoblog”–just some fun pictures from our notebooks.
    The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!
The brightest cyan, green fluorescent proteins, and the brightest 
ever FP in LanYFP!
Ain't they pretty?
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.
2-3 times brighter than EGFP, no cytotoxicity detected
The mWasabi is stimulating
The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.
    The LanFPs express well in bacteria.
Reminding you of icecream
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.
Fluorescence while running in denaturing gel
Can you see the FP bands in the SDS PAGE?
    FPs in SDS PAGE–a closer look
while the gel is still running
Can you see them now?
    FPs in gel cassette over UV lights
Easier to see now than during gel running
Invincible FPs
    FPs in gel cassette under blue LED
The red FP is harder to see because of the black background
Fluorescence in SDS page 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.

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.

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.