Expanding the Camelid Antibody Product Line

While Chromotek GFP-Trap resin has become one of the best sellers from the Allele Biotech’ Camelid Antibody (VHH antibody) product line, more products have been added that will prove to be great tools for GFP-related research.

GFP is a powerful tool to study protein localization and dynamics in living cells. However, the photo stability and the quantum efficiency of GFP are not sufficient for Super-Resolution Microscopy (e.g. 3D-SIM or STED) of fixed samples from cells expressing GFP-fusion proteins to visualize specific structures. Furthermore, many cell biological methods such as HCl treatment for BrdU-detection, the EdU-Click-iT™ treatment or heat denaturation for FISH lead to disruption of GFP signal.

Now we offer our GFP-Trap Booster for reactivation, boosting and stabilization of GFP, suitable for acquiring strong and long lasting signals from GFP-fusion proteins. It is based on a specific GFP-binding protein as in GFP-Trap but coupled to the fluorescent dye ATTO 488 (from ATTO-TEC). For information, please read the product description of this week New Product of the Week: GFP-Trap booster, ABP-CM-GBOOSTR, http://www.allelebiotech.com/shopcart/index.php?c=221&sc=158

Promotion of the week: All mTFP1 and mWasabi fusion plasmids are 30% off for this week only

Preview of future new product: a similarly high quality product, the RFP-Trap that pulls down DsRed derived proteins including mRFP1, mCherry, mOrange, mPlum but also mRuby and RFP-tagged fusion proteins.

Tags: anti-GFP, Chromotek, GFP coIP, GFP-Trap, mCherry, mOrange, mPlum, mRFP1, mRuby RFP-tagged fusion proteins, nti-RFP, pulldown, RFP-Trap, VHH antibody

Anti-GFP antibody choices

A variety of anti-GFP antibodies are now provided to fluorescent protein users. In addition to the monoclonal anti-GFP antibody that has been introduced together with the GFP-Trap product group, Allele Biotech also has a strong anti-GFP polyclonal product, and a new rat anti-GFP monoclonal antibody. The availability of these different species specificity should allow users to have options in staining, detection, co-IP, double-labeling, etc.

New Product of the Week 04-05-10 to 04-11-10: Rat anti-AvGFP antibody, ACT-CM-MRGFP10

Promotion of the Week 04-05-10 to 04-11-10: Buy any Allele Biotech/Orbigen’s polyclonal antibody, get another of equal or less value at 60% off (if you are not a fan or friend through Allele’s online social networks, the discount is 30%).

Tags: 3H9, anti-GFP, antibodies, camelid antibodies, Chromotek, GFP booster, GFP detection, monoclonal, polyclonal, rat monoclonal

Using 2A “self-cleaving” peptide in bicistronic mammalian expression

Multiple promoters or internal ribosomal entry sites (IRES) have been used for the production of multiple proteins from the same vector. Potential drawbacks with multiple promoters on viral vectors include unstable genome and interference between promoters. IRES is a relatively large sequence that can cause problems in virus packaging, especially for viruses with very limited genome size such as AAV. In addition, it is required that the start of the second ORF is fairly close to the IRES, adding difficulties to cloning.

2A or 2A-like peptide (collectively called 2A peptide here) is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. Although called a “self-cleaving” peptide or protease site, the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping–a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation even.

The 2A-based bicistronic expression has been used for several years, but recently gained much more popularity due to its successful use in iPSC generation that required 2 to 4 factors working in concert. Even expression of all factors can be achieved when 2A peptides are used for multiple protein production, due to near 100% efficiency of the 2A “cleavage” at each site, and no interference between multiple 2A sites. Early work used a 36 amino acid sequence as 2A peptide, which was later reduced to about half that size from mutation and screening. Commercial vectors utilizing 2A for co-expression of cDNA and fluorescent protein and/or drug resistance genes have not been available until now. Allele Biotech has introduced a number of such plasmids, establishing another First-to-the-market as it has done many times previously in its 10 year history.

New product of the week 03-29-10 to 04-04-10:

Alleleustrious pmTFP1-2A Bicistronic mammalian expression vector, ABP-FP-T2A10, $399, http://www.allelebiotech.com/shopcart/index.php?c=215&sc=34

Promotion of the week 03-29-10 to 04-04-10:

Buy any GFP-Trap beads or kits, get polyclonal anti-GFP (ABP-PAB-PAGFP10) at half size for FREE!  ***GFP-Trap has been replaced with GFP-nAb, an improved version for the same purposes in the form of agarose beads, magnetic beads, polyacrylamide beads, spin column kits, etc.  Come and take a look at the most comprehensive list of nearly all FPs before purchasing.

Tags: 2A peptide, anti-GFP, bicistronic, drug resistance, GFP-Trap, iPS, iPSCs, IRES, polycistronic, ribosome skip, self-cleaving, transcription, translation

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

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.

Tags: cell imaging, CFP, FLIM-FRET, Fluorescent proteins, FP, FRET, FRET acceptor, FRET donor, FRET pair, GFP, GFP fusion, mTFP1, mWasabi, subcellular localization