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.
Thursday, June 24, 2010
Saturday, June 19, 2010
From Nature Biotechnology: SBIR grants wax
Awards under the Small Business Innovation Research (SBIR) program have just been given a boost. As of March 30, the cap for SBIR phase I awards has risen from $100,000 to $150,000, and for phase II awards from $750,000 to $1,000,000. The increases are intended to take account of inflation since 1992 when the threshold amounts were last set by Congress. "This will have an important positive impact at a critical [juncture] in the aftermath of the nation's great recession," says Simcha Jong, university lecturer in management science and innovation at University College London. Jong says that, historically, the SBIR program helped forge links between university science and industry and, at this pivotal time, could help kick-start the US job engine. The Senate has passed a bill to extend the SBIR and related Small Business Technology Transfer through July 31 (Nat. Biotechnol. 27, 1065-1066, 2009). Even more generous than SBIR grants are the new Small Business Helping Investigators to Fuel the Translation of Scientific Discoveries (SHIFT) awards launched on March 5 by the US Department of Health and Human Services. These awards, aimed at fostering translational research, offer companies up to $2.65 million over five years. "The main point is to encourage current academic researchers to apply, and use it to move to biotech," says Jiwu Wang, president and CEO of Allele, a San Diego-based company that has taken products to market with SBIR support. "It is a great idea."
Emma Dorey, Nature Biotechnology
Emma Dorey, Nature Biotechnology
Sunday, June 13, 2010
Oct3/4 Promoter Blocks Cancer Stem Cells from Differentiation—Potential for Stably Supplying CSCs
Cancer stem cells (CSCs) are thought to be a group of cells within tumor tissues that are resistant to standard chemotherapy and the main cause for relapse. Typically identified by cell surface markers (e.g. CD44, CD24), CSC cells belong to a minority population among cancer cells, which are difficult to obtain or maintain. There have been several NIH programs soliciting novel methods of isolating CSCs for functional studies and screen for therapies.
Sajithlal et al. reported in Stem Cells that by introducing an Oct3/4 promoter-GFP construct in an attempt to follow CSCs among MCF7 breast cancer cells, they unexpectedly found out that this construct blocked differentiation of CSCs and maintained their signature cell surface marker expression profile. It is not easy to explain how a promoter (assuming the expressed GFP has nothing to do with the effects of this construct) would block differentiation, however, the authors did speculate that it could be some ORFs or small RNAs encoded within the 4 kb promoter region, or its competition with endogenous promoter. They went on to test the same construct in several other breast cancer cell lines and found similar results. It remains unclear whether it would be true in other cancer types, isolated cancer cells instead of cell lines, or if other stem cell specific promoters, such as Nanog promoter, would have similar effects.
For those who are interested in CSCs, this new finding should be of great interest for theoretical as well as practical reasons.
STEM CELLS 2010;28:1008–1018 www.StemCells.com, photo courtesy
AlleleNews JW
Related product from Allele Biotech iPS product line; Pluripotency reporter lentivirus, ready-to-use , e.g. Validated Oct-4 Promoter Reporter Lentiviral Particles, 5 vials ABP-SC-ROCT4G5 $450.00.
Sajithlal et al. reported in Stem Cells that by introducing an Oct3/4 promoter-GFP construct in an attempt to follow CSCs among MCF7 breast cancer cells, they unexpectedly found out that this construct blocked differentiation of CSCs and maintained their signature cell surface marker expression profile. It is not easy to explain how a promoter (assuming the expressed GFP has nothing to do with the effects of this construct) would block differentiation, however, the authors did speculate that it could be some ORFs or small RNAs encoded within the 4 kb promoter region, or its competition with endogenous promoter. They went on to test the same construct in several other breast cancer cell lines and found similar results. It remains unclear whether it would be true in other cancer types, isolated cancer cells instead of cell lines, or if other stem cell specific promoters, such as Nanog promoter, would have similar effects.
For those who are interested in CSCs, this new finding should be of great interest for theoretical as well as practical reasons.
STEM CELLS 2010;28:1008–1018 www.StemCells.com, photo courtesy
AlleleNews JW
Related product from Allele Biotech iPS product line; Pluripotency reporter lentivirus, ready-to-use , e.g. Validated Oct-4 Promoter Reporter Lentiviral Particles, 5 vials ABP-SC-ROCT4G5 $450.00.
Monday, June 7, 2010
Introducing Product-on-Demand Biological Research Reagents
http://allelebiotech.com/blogs/2010/06/introducing-product-on-demand-not-going-to-be-called-ipod-biological-research-reagents/
The general order of operations in the bioreagent industry begins with a developer observing or forecasting a need and developing a product. The supplier then supplies that product to customers by showing that the product will suit their existing needs. An alternative order in our industry is after a new discovery in the form an enzyme reaction mechanism, affinity binding, or biological system is made in lab, someone realizes that discovery could be made into a product. If the idea is picked up by a commercial R&D team, the underlining mechanisms of the discovery are then exploited for particular use and reagents or kits will be built around it. The new products are introduced to the market by convincing potential users that they will make their research better, cheaper, or faster.
From a supplier’s point of view, if the current processes for developing new products have been working, what’s the incentive to change? From a researcher’s point of view, well, do they have any other choices? If something is not commercially available, someone will just make it in the lab if they need it. Some of us still remember the days when a graduate student needed to make his own restriction enzyme because NEB didn’t sell it. However, there is a disconnect between how much new knowledge is being gained every single day in tens of thousands of labs and how small a portion of that knowledge pool is being turned into more powerful tools to make the next round of research easier and more cost-effective. For instance, when an important gene’s promoter is recently defined by a functional study in 293T cells, how soon do you expect to test the signals that influence transcription from that promoter in the primary cells you are working on? Wouldn’t it be nice if you could simply buy a vector that will express a promoter-driven reporter ready to be introduced into the primary cells in your lab instead of having a graduate student design, construct, learn and try to make a lentiviral vector in the next few months?
And yes, there is the route called custom projects provided by a few bioreagent companies. The prices are often inhibiting for the reasons that the price needs to cover for labor on industry pay scale, materials, indirect, and profit. Additionally, since the service provider does not take ownership of the product, the work of researching the relevant pathways and making construct designs is left to the user.
There is a better way. A company can plan product groups, lines, and packages based solely on the demonstrated importance of a system such as signal pathway or a family of molecules like miRNA. The plan can project to use the most advanced technologies, even accompanied with full product descriptions and vector maps. However, it would be a great waste of money and material if nobody would ever need it, right? One way of dealing with the initial cost is that we make the first kit upon the first order. The customer that places the first order of a new product will get a deep discount off the shelf-product price on what used to be a custom project. They might even have the opportunity to provide input on the product design prior to production. From a supplier side, we will benefit by having an opportunity to initiate a new product without major investment, which in turn would keep our overall prices low for such innovative and advanced products.
This model should help speed up the commercial application of any new biological findings, lower the cost and price of bioreagent products, and encourage interaction between researchers who normally do not work with each other to produce better products for increasing the efficiency of research.
Discount of the week 060110-060710: Any virus packaging project initiated this week gets additional 10% discount that can be used with first time discount and other pricing advantages. http://www.allelebiotech.com/allele3/Services_Lentiviral_Retroviral_Packaging.php
New product of the week 060110-060710: Columns for Miniprep and Gel Purification, ABP-PP-COLM100. If you can make your own buffers or have leftovers from any miniprep or gel purification kits, get these high capacity columns and lower your costs by up to 70%!
The general order of operations in the bioreagent industry begins with a developer observing or forecasting a need and developing a product. The supplier then supplies that product to customers by showing that the product will suit their existing needs. An alternative order in our industry is after a new discovery in the form an enzyme reaction mechanism, affinity binding, or biological system is made in lab, someone realizes that discovery could be made into a product. If the idea is picked up by a commercial R&D team, the underlining mechanisms of the discovery are then exploited for particular use and reagents or kits will be built around it. The new products are introduced to the market by convincing potential users that they will make their research better, cheaper, or faster.
From a supplier’s point of view, if the current processes for developing new products have been working, what’s the incentive to change? From a researcher’s point of view, well, do they have any other choices? If something is not commercially available, someone will just make it in the lab if they need it. Some of us still remember the days when a graduate student needed to make his own restriction enzyme because NEB didn’t sell it. However, there is a disconnect between how much new knowledge is being gained every single day in tens of thousands of labs and how small a portion of that knowledge pool is being turned into more powerful tools to make the next round of research easier and more cost-effective. For instance, when an important gene’s promoter is recently defined by a functional study in 293T cells, how soon do you expect to test the signals that influence transcription from that promoter in the primary cells you are working on? Wouldn’t it be nice if you could simply buy a vector that will express a promoter-driven reporter ready to be introduced into the primary cells in your lab instead of having a graduate student design, construct, learn and try to make a lentiviral vector in the next few months?
And yes, there is the route called custom projects provided by a few bioreagent companies. The prices are often inhibiting for the reasons that the price needs to cover for labor on industry pay scale, materials, indirect, and profit. Additionally, since the service provider does not take ownership of the product, the work of researching the relevant pathways and making construct designs is left to the user.
There is a better way. A company can plan product groups, lines, and packages based solely on the demonstrated importance of a system such as signal pathway or a family of molecules like miRNA. The plan can project to use the most advanced technologies, even accompanied with full product descriptions and vector maps. However, it would be a great waste of money and material if nobody would ever need it, right? One way of dealing with the initial cost is that we make the first kit upon the first order. The customer that places the first order of a new product will get a deep discount off the shelf-product price on what used to be a custom project. They might even have the opportunity to provide input on the product design prior to production. From a supplier side, we will benefit by having an opportunity to initiate a new product without major investment, which in turn would keep our overall prices low for such innovative and advanced products.
This model should help speed up the commercial application of any new biological findings, lower the cost and price of bioreagent products, and encourage interaction between researchers who normally do not work with each other to produce better products for increasing the efficiency of research.
Discount of the week 060110-060710: Any virus packaging project initiated this week gets additional 10% discount that can be used with first time discount and other pricing advantages. http://www.allelebiotech.com/allele3/Services_Lentiviral_Retroviral_Packaging.php
New product of the week 060110-060710: Columns for Miniprep and Gel Purification, ABP-PP-COLM100. If you can make your own buffers or have leftovers from any miniprep or gel purification kits, get these high capacity columns and lower your costs by up to 70%!
Friday, June 4, 2010
If you were there
Tell the world, tell those next to you, what happened 21 years ago. Enjoy what you have, do what you do, but lead an honorable life worthy of those lost in the fight for freedom.
Monday, May 31, 2010
Saturday, May 29, 2010
Lentiviruses expressing constitutively active or dominant negative versions of genes in signaling pathways
NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) refers to a protein complex functional in signaling pathways, particularly in response to stress stimuli. There are two signaling pathways leading to the activation of NF-kB signaling, known as the canonical (or classical) pathway, the non-canonical (or alternative) pathway. (from AlleleBlog)
In the canonical NF-kB pathway, NF-kB dimers such as p50/RelA are maintained in the cytoplasm by interaction with an independent Inhibitor of NF-kB (IkB) molecule. When the upstream signaling is active, an IkBa kinase (IKK) complex consisting of catalytic kinase subunits IKKa and/or IKKb and the scaffold protein NEMO will be recruited to the cytoplasmic adaptor of certain cell surface receptor and stay activated. Activation of IKK complex will consequently phosphorylate the IkB at two serine residues, which induce the proteasomal degradation of IkB. Released from IkB, NF-kB dimers then translocate into the nucleus and bind with a consensus sequence (GGGACTTTCC) of various genes and thereby activates their transcription. In a negative feedback loop, NF-kB activation also leads to the expression of the IkB gene, which subsequently sequesters NF-kB subunits and terminates transcriptional activity unless a constitutive activation signal is present.
The non-canonical pathway is mainly for activation of p100/RelB complexes during B-and T-cell development, where NF-kB was first discovered. Different from the canonical pathway, 1) only certain receptor signals (e.g., Lymphotoxin B, B-cell activating factor, CD40) can activate this pathway, 2) it proceeds through an IKK complex that contains two IKKa subunits (but not NEMO) and 3) receptor binding leads to activation of the NF-kB-inducing kinase NIK, which phosphorylates and activates an IKKa complex, the latter in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal IkB domain of p100, leading to its partial proteolysis and liberation of the p52/RelB complex.
Other distinct NF-kB pathways undoubtedly exist. For example, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction with the IkB-like co-activator Bcl-3 (or IkBz). How these are regulated is not known.
There are many ways of applying reagents that can manipulate the NF-kB system for drug screening as well as basic research. For this reason, Allele Biotech is introducing a set of lentiviruses as listed below. The way this product group is operated is that the Allele will publish the design and specifics of the products, but will produce it upon the first order. The person who places the first order will need to wait for 3-5 weeks for receiving the products, but will receive a 40% discount and an opportunity to provide input to the product design. The platform for lentiviral products is based on our field-leading high titer lentivirus packaging capabilities, and you can rest assured that high quality lentivirus will be produced at a pace much faster and lower price than if you were to make them by your own lab or find them from anywhere else. Allele Biotech will introduce more such products in the fields of miRNA (to provide all ~800 human miRNA and their anti-miRNA silencers on virus), cell differentiation lineage-specific promoter-FP reporters, etc. We now call these products the Lead Products. Cost effectiveness is just the beginning. Imagine how our ingenuity and innovation can pave the way for your research.
1) HiTiter IkBa Expression Lentiviral Particles: can be used in functional studies of IkBa’s roles in NF-kB signaling. Overexpression of IkBa will eliminate the low-level activation by factors in the cell culture medium or other fluctuations to ensure that any change in NF-kB signaling is caused by the stimulus being tested.
2) HiTiter Dominant Negative IkBa Expression Lentiviral Particles: Dominant Negative IkBa has serine-to-alanine changes at residues 32 and 36. It can be used to repress the expression of endogenous IkBa or block NFkB signaling in certain cell lines.
3) HiTiter IkBa-RFP Fusion Expression Lentiviral Particles: This is a reporter suitable for the studying proteasomal degradation of IkBa after phosphorylation.
4) HiTiter IKKa Expression Lentiviral Particles: can be used in functional research for the role of IKKa in NF-kB signaling.
5) HiTiter Constitutively Active IKKa Expression Lentiviral Particles: Constitutively active IKKa has serine-to-glutamate mutations at residues 176 and 180.
6) HiTiter Dominant Negative IKKa Expression Lentiviral Particles: Dominant negative IKKa is mutated by serine-to-alanine at residues 176 and 180.
7) HiTiter IKKb Expression Lentiviral Particles: can be used in functional research for the role of IKKb in NF-kB signaling.
8) HiTiter Constitutively Active IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-glutamate at residues 177 and 181.
9) HiTiter Dominant Negative IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-alanine at residues 177 and 181.
10) HiTiter pNFkB-Luciferase Lentiviral Particles: a cis-reporter plasmid containing the luciferase reporter cDNA linked to five repeats of an NF-kB binding site.
11) HiTiter pNFkB-GFP Lentiviral Particles: a cis-reporter plasmid containing the GFP reporter gene linked to five repeats of an NF-kB binding site.
New Product of the Week 05-23-10 to 05-31-10:
Cre recombinase fused to mWasabi GFP carried on lentivirus, ABP-RP-Cre2AGL, or ABP-RP-Cre2AGS
Promotion of the week 05-23-10 to 05-31-10:
actually 3 promotions this week, first announced to Allele fans and friends on Facebook: 10% off the brand-new RFP-Trap ACT-CM-RFA0050 for co-IP with mCherry, mPlum, mOrange…Order by Monday to qualify for discount! Free sample for FAM-amidite for oligo cores at universities that make qPCR and other probes. Free sample of high attachment tissue culture plates that use 40% less plastics (made in Canada)
In the canonical NF-kB pathway, NF-kB dimers such as p50/RelA are maintained in the cytoplasm by interaction with an independent Inhibitor of NF-kB (IkB) molecule. When the upstream signaling is active, an IkBa kinase (IKK) complex consisting of catalytic kinase subunits IKKa and/or IKKb and the scaffold protein NEMO will be recruited to the cytoplasmic adaptor of certain cell surface receptor and stay activated. Activation of IKK complex will consequently phosphorylate the IkB at two serine residues, which induce the proteasomal degradation of IkB. Released from IkB, NF-kB dimers then translocate into the nucleus and bind with a consensus sequence (GGGACTTTCC) of various genes and thereby activates their transcription. In a negative feedback loop, NF-kB activation also leads to the expression of the IkB gene, which subsequently sequesters NF-kB subunits and terminates transcriptional activity unless a constitutive activation signal is present.
The non-canonical pathway is mainly for activation of p100/RelB complexes during B-and T-cell development, where NF-kB was first discovered. Different from the canonical pathway, 1) only certain receptor signals (e.g., Lymphotoxin B, B-cell activating factor, CD40) can activate this pathway, 2) it proceeds through an IKK complex that contains two IKKa subunits (but not NEMO) and 3) receptor binding leads to activation of the NF-kB-inducing kinase NIK, which phosphorylates and activates an IKKa complex, the latter in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal IkB domain of p100, leading to its partial proteolysis and liberation of the p52/RelB complex.
Other distinct NF-kB pathways undoubtedly exist. For example, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction with the IkB-like co-activator Bcl-3 (or IkBz). How these are regulated is not known.
There are many ways of applying reagents that can manipulate the NF-kB system for drug screening as well as basic research. For this reason, Allele Biotech is introducing a set of lentiviruses as listed below. The way this product group is operated is that the Allele will publish the design and specifics of the products, but will produce it upon the first order. The person who places the first order will need to wait for 3-5 weeks for receiving the products, but will receive a 40% discount and an opportunity to provide input to the product design. The platform for lentiviral products is based on our field-leading high titer lentivirus packaging capabilities, and you can rest assured that high quality lentivirus will be produced at a pace much faster and lower price than if you were to make them by your own lab or find them from anywhere else. Allele Biotech will introduce more such products in the fields of miRNA (to provide all ~800 human miRNA and their anti-miRNA silencers on virus), cell differentiation lineage-specific promoter-FP reporters, etc. We now call these products the Lead Products. Cost effectiveness is just the beginning. Imagine how our ingenuity and innovation can pave the way for your research.
1) HiTiter IkBa Expression Lentiviral Particles: can be used in functional studies of IkBa’s roles in NF-kB signaling. Overexpression of IkBa will eliminate the low-level activation by factors in the cell culture medium or other fluctuations to ensure that any change in NF-kB signaling is caused by the stimulus being tested.
2) HiTiter Dominant Negative IkBa Expression Lentiviral Particles: Dominant Negative IkBa has serine-to-alanine changes at residues 32 and 36. It can be used to repress the expression of endogenous IkBa or block NFkB signaling in certain cell lines.
3) HiTiter IkBa-RFP Fusion Expression Lentiviral Particles: This is a reporter suitable for the studying proteasomal degradation of IkBa after phosphorylation.
4) HiTiter IKKa Expression Lentiviral Particles: can be used in functional research for the role of IKKa in NF-kB signaling.
5) HiTiter Constitutively Active IKKa Expression Lentiviral Particles: Constitutively active IKKa has serine-to-glutamate mutations at residues 176 and 180.
6) HiTiter Dominant Negative IKKa Expression Lentiviral Particles: Dominant negative IKKa is mutated by serine-to-alanine at residues 176 and 180.
7) HiTiter IKKb Expression Lentiviral Particles: can be used in functional research for the role of IKKb in NF-kB signaling.
8) HiTiter Constitutively Active IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-glutamate at residues 177 and 181.
9) HiTiter Dominant Negative IKKb Expression Lentiviral Particles: Dominant negative IKKb is mutated by serine-to-alanine at residues 177 and 181.
10) HiTiter pNFkB-Luciferase Lentiviral Particles: a cis-reporter plasmid containing the luciferase reporter cDNA linked to five repeats of an NF-kB binding site.
11) HiTiter pNFkB-GFP Lentiviral Particles: a cis-reporter plasmid containing the GFP reporter gene linked to five repeats of an NF-kB binding site.
New Product of the Week 05-23-10 to 05-31-10:
Cre recombinase fused to mWasabi GFP carried on lentivirus, ABP-RP-Cre2AGL, or ABP-RP-Cre2AGS
Promotion of the week 05-23-10 to 05-31-10:
actually 3 promotions this week, first announced to Allele fans and friends on Facebook: 10% off the brand-new RFP-Trap ACT-CM-RFA0050 for co-IP with mCherry, mPlum, mOrange…Order by Monday to qualify for discount! Free sample for FAM-amidite for oligo cores at universities that make qPCR and other probes. Free sample of high attachment tissue culture plates that use 40% less plastics (made in Canada)
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