Allele’s 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!

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.

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.

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.

Can you see the FP bands in the SDS PAGE?

FPs in SDS PAGE–a closer look

Can you see them now?

FPs in gel cassette over UV lights

Invincible FPs

FPs in gel cassette under blue LED

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

Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures

Allele Custom Services for Drug Screening Companies

Many target discovery and validation programs can benefit from RNA interference, fluorescent proteins, stem cells, and viral delivery systems. However, applications of these technologies require special reagents and laboratory know-how. Even when available, many generic reagent kits are not tailored for your particular needs in screening or validation.

At Allele, we accelerate your discovery efforts with custom RNAi screening, fluorescence based assays, and cell model development services.

1) Our RNAi platform, based on our patented shRNA/miRNA technologies, use DNA linear template, plasmid, lentivirus, retrovirus, or baculovirus vectors that prompt cells to endogenously express RNAi. As a result, our screens offer advantages over synthetic siRNAs:
• Higher levels of consistency
• Greater delivery and gene silencing efficiencies
• Accessibility to difficult-to-transfect cells, including primary cells
• Potential for inducible RNAi expression
• More persistent silencing with shRNA under Allele’s own IP–you may not need to license siRNA patents!

2) Fluorescent proteins (FPs), which can span the entire visual spectrum, have become some of the most widely used genetically encoded tags. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Allele Biotech is one of a few companies that develop and improve FPs through fundamental research. We have so far achieved:
• The brightest cyan and green FPs, true monomers for minimum artifact or cytotoxicity
• The brightest yellow and red FPs from lancelet, only FPs from vertebrate
• mTFP1 as the best FRET donor by 3 independent reports
• Photoconvertible FPs for super imaging or kinetic labeling
• Delivery on plasmid, retrovirus, lentivirus, baculovirus vectors

3) As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors. These induced stem cells (iPSCs) provide unprecedented resources of cells of different differentiation stages for functional testing and drug screening. Allele Biotech develops and provides state-of-the-art reagents in convenient forms for iPSC production
• iPS factors carried on lentivirus, retrovirus, baculovirus for different cell types
• Availability in combination with fluorescent proteins under own IP, and drug resistant genes
• 4-in-1 or 2-in-1 effective use of iPS factors on one viral vector
• Feeder cells of human origin expressing factors essential for stem cell culturing

4) Introduction of protein factors, miRNA, promoter-reporter, and virtually any other genetic element of interest via the most efficient viral packaging systems.
• Introducing protein-FP fusion, promoter-FP reporter, photoactivatable factors for cell-based assays
• Introducing critical factors for cell immortalization
• Episomal or integrated expression using baculoviral vectors
• High throughput, systematic expression of whole class of molecules in any type of cell
• High titer viral packaging at low cost for delivery to animal tissues

In addition, the Allele team can provide custom-designed assays that can be used for assaying enzyme activities in almost any pathway, such as the EGF pathway, TNF response/apoptosis pathway, nuclear receptors, etc. We utilize technically advanced methods to provide our partners with advantages over alternative methods or other services.

New Product of the Week 06-28-10 to 07-03-10: Eco-friendly mammalian tissue culture plates, 40% less plastic to the environment, 40% less cost to your budget, contact our sales rep today for quotes and details.

Promotion of the Week 06-28-10 to 07-03-10: Oct3/4 iPS lentivirus with RFP as marker, new to the market, this week only all kits containing Oct3/4-RFP same price as the original, non-RFP versions, save ~$50!

Tags: cell assays, cell based assays, cell culture, drug screening, FP assays, FRET, iPS cells, kinase assasy, pathway, RNAi

Developing Cell-Based Assays

Cascaded protein interactions form the foundation of all signaling pathways, many of which are involved in multiple human diseases. These interactions are sensitively and precisely regulated by various post-translation modifications such as phosphorylation, acetylation, ubiquitination, etc. Many action points of the protein modifications have been targeted during drug development. From a survey we have conducted on assays aimed at these targets, we found that most of the assays are based on the enzymatic reactions, e.g. phosphorylation-specific ELISA, and chemically modified FRET, which require pre-assembled reagent kits which are hard to apply to different targets and different cell models.

Fluorescent Protein based FRET might be the optimal choice to develop a versatile cell-based assay. Since signaling pathways rely on hierarchical protein-protein interactions, the most direct and precise way to study cell signaling pathways would be to detect the interactions between a target protein and its immediate downstream protein. Furthermore, different upstream signals can activate the same set of target proteins in different post-modification patterns, resulting in specific activation of downstream responding factors. These signal flows may be individually monitored by using FRET based assay redesigned and validated for each downstream pathway.

Allele’s scientists can develop cell-based assays with in-depth understanding of protein interactions within the context of human genome, such as the SH2, SH3 and PTB domains in tyrosine kinase signaling, the F-box, BTB-box, SOCS, WDR, and LRR domains in the ubiquitin proteasome system, etc. Additionally, Allele’s cell-based assays can be carried on world’s most powerful lentivirus packaging platform, suitable for virtually all different cell lines and primary cells.

New Product of the Week 061410 to 062010: Rat monoclonal antibody against GFP, strong signal for GFP labeling

Promotion of the Week 061410 to 062010: Mouse LIF-bFGF expressing feeder cells for stem cell culture (ABP-SC-BLIFM05), one vial free to go with any iPS lentivirus/retrovirus kit.

Tags: BTB-box, cell based assays, F-box, FRET, LRR domain, pathway, signaling pathway, SOCS, WDR domain

Fluorescent Protein-Based Assay Development II

FPs as pH and redox sensors:

The uses of FPs extend well beyond simple expression and fusion reporters.  While pH sensitivity (usually quenching of fluorescence by acidic pH) is generally considered a drawback for fusion tagging, it becomes a useful property for constructing pH sensors.  FPs specifically engineered to take advantage of pH sensitivity (“pHluorins”) report pH as either a change in fluorescent intensity or a change in the ratio of excitation at two different wavelengths, and may be used to monitor processes such as endocytosis or other pH-variable processes.  In such an application, the pH-sensitive FP is fused to a localization tag for the compartment of interest which experiences variable pH.  This technique can be used, for example, to visualize release of neurotransmitter-containing vesicles.  In addition to pH-sensitive FPs, redox-sensitive Aequorea GFP variants have been produced (roGFPs and others) which produce similar changes in fluorescence intensity or excitation ratio when exposed to differing redox conditions or reactive oxygen species.

Sensors based on circularly permuted FPs:

Because FPs have such a compact and stable beta-barrel fold with N and C termini close together, it is possible to engineer circularly permuted variants which retain their fluorescent properties.  Studies on circular permutation of FPs have led to the development of several different sensors which take advantage of domains inserted into sensitive areas of the fluorescent protein backbone.  The most famous of these are the GCaMP calcium sensors, in which a calmodulin domain has been inserted into a loop in GFP, yielding a sensor that reports calcium concentration as a change in fluorescent intensity.  Other circularly permuted FP variants, such as cpVenus (a yellow Aequorea GFP variant), have found usefulness in improving FRET sensor dynamic range (see next section).

FRET sensors:

Fluorescence resonance energy transfer (FRET) is a quantum mechanical process that allows the transfer of excited state energy between two fluorophores when they are in close physical proximity.  Because this process operates with a strong distance (1/r^6) and orientation dependence (strongest when chromophore dipoles are parallel or antiparallel), it lends itself to the construction of highly sensitive reporters of biochemical activity.  In FP FRET, excited state energy from a higher-energy (shorter wavelength) “donor” fluorescent protein is transferred to a lower-energy (longer wavelength) “acceptor” FP, leading to sensitized fluorescent emission from the acceptor and reduced emission (quenching) from the donor.  By linking donor and acceptor FPs with a domain which changes conformation in response to a biochemical activity of interest, this activity is reported as a change in the ratio of sensitized emission to direct-excitation emission of the acceptor (or a simple ratio of donor and acceptor emission).  FRET sensors have been engineered to specifically sense a wide variety of activities, including many protein kinases, as well as small molecules such as Ca2+ and neurotransmitters.  While design of a new FRET sensor generally requires a great deal of optimization and trail-and-error, this class of probe is among the most powerful tools currently available for investigating live-cell biochemistry.

New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.

Tags: animal models, biosensors, calmodulin, cell based assays, circular permutatioon, Fluorescent proteins, FP sensor, FRET, GCaMP, pH sensors, redox sensor

Fluorescent Protein-Based Assay Development

This blog is a preview of what is to be launched as a new Service Group. Allele Biotech is restructuring its CRO capabilities in the assay development area by combining its fast expanding fluorescent protein portfolio, viral vector and packaging expertise, as well as newly granted patents in shRNA. The focus of this post is fluorescent protein in biosensor and screening assays. A modified version will be used as the landing page for the FB-Based Assay Development Service.

Overview:

Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.

Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.

FPs as fusion tags:

Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.

FPs as expression reporters:

FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.

New Product of the Week 03-08-10 to 03-14-10: mWasabi 2A or IRES dual expression vectors (http://www.allelebiotech.com/shopcart/index.php?c=216&sc=34) ABP-FP-W2A10, orWIRES10

Promotion of the Week 03-08-10 to 03-14-10: for a limited time on Thursday, to be announced on our Facebook page (http://www.facebook.com/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957#!/allele.biotech?ref=profile), a strikingly low price will be honored for a commonly used lab reagent or equipment. This is the second week of the follow-us-to-the-basement promotion.

Tags: assay development, biosensor, cell based assays, chemical screening, circular permutation, fluorescent protein, FRET, GFP, mTFP1, mWasabi, promoter-FP, RNAi screening