Camelid Antibody
17 Papers Using GFP-Trap, 12 Since 2009
1. MacKay C, Déclais AC, Lundin C et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142:65-76.
2. Babiano R, de la Cruz J. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 2010 Apr 14.
3. Fulcher AJ, Dias MM, Jans DA. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem. 285:17744-53.
4. Taniue K, Nishida A, Hamada F et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development. 137:1755-64.
5. Rottach A, Frauer C, Pichler G et al. (2010). The multi-domain protein Np95 connects DNA methylation and histone modification. Nucleic Acids Res. 38:1796-804.
6. Boulon S, Ahmad Y, Trinkle-Mulcahy L et al. (2010). Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9:861-79.
7. Schornack S, Fuchs R, Huitema E et al. (2009). Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60:744-54.
8. Fellinger K, Bultmann S, Rothbauer U et al. (2009). Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells. EMBO Rep. 10:1259-64.
9. Muñoz IM, Hain K, Déclais AC et al. (2009). Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol Cell. 35:116-27.
10. Webby CJ, Wolf A, et al. (2009). Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing. Science. 325:90-93.
11. Rogowski K et al. (2009). Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell. 137: 1076-87.
12. Frauer C, Leonhardt H, (2009) A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding. Nucleic Acid Res. 35: 5402-5409.
13. Trinkle-Mulcahy L et al., (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183: s223-39.
14. Rothbauer U, Leonhardt H, (2008) Connecting Biochemistry and Cell Biology with Nanobodies. Zellbiologie aktuell 34: 9-12.
15. Rothbauer U et al., (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282-289.
16. Agarwal N et al., (2007) MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. Nucleic Acid Res.35: 5402-5409.
17. Rothbauer U et al., (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3: 887-889.
New Product of the Week 071910-072510: Cre Reporter Cell Line: LoxP-RFP Human Fibroblast, perfect to test our Cre-2A-GFP lentivirus, when cre works, the cell change from red to green.
Promotion of the Week 071910-072510: High Quality dNTP Mix, 10mM, 5 ml, $409 this week $309. Hurry, email to oligo@allelebiotech.com or fax 858-587-6692 by Sunday to save $100 on your lab budget.
BioTechniques Publishes Article on Single Domain Antibodies
Many blogs start by asking “Did you know…” to intrigue you to read along. So here it goes:
Did you know that there are more than 300,000 antibodies that are commercially available? And yes, many antibody companies are still generating more antibodies at ever faster pace and in a more systematic way. There are companies that plan to make peptide or short protein fragments for making antibodies against all human proteins or subproteome, others develop antibodies particularly suitable for demanding assays such as ChIP-CHIP. Government activities such as the National Cancer Institute (NCI)’s Clinical Proteomic Technologies Initiative (CPTI) and the Road Map program under the NIH Director’s Office also set goals of producing comprehensive sets of widely usable, renewable, affinity reagents for clinical cancer samples or the human proteome. Apparently people do not think the 300,000 available antibodies are sufficient for what they do.
Did you know that conventional antibodies commonly used as reagents are ~150kDa in molecular weight and can hardly be used inside live cells? Ulrich Rothbauer, professor in the department of biology at Ludwig Maximilians University, who is working with colleagues to develop tools to study cellular processes in living cells. “These antibodies have to assemble four different chains, two heavy and two light, and they’re assembled by disulfide bonds that cannot be correctly formed in the reducing environment of the cytoplasm. You cannot express such a huge complex molecule in living cells. You can [introduce] them by microinjection, for example, but it’s not applicable for high-throughput cell imaging.” [1] Antibody fragments such as scFv, Fab, and similar derivatives have been developed over the years to certain level of success, but not as widely accepted or practically amenable to replacing conventional antibodies.
Did you know that camel, llama, and shark naturally produce single heavy chain antibodies that can function as 13-16kDa fragments (yes if you have read previous Allele Blogs http://allelebiotech.com/blogs/2009/08/camelid-antibodies/)? They can easily be produced in bacteria, used directly inside live cells via transgene, fused to other proteins as a fusion tag, linked to DNA oligos as a detection module, or immobilized on beads for pull down or co-IP. Currently, these antibodies need to be selected by display after obtaining immunized antibody libraries. There is generally no commercial service for creating custom camelid antibodies at this time due to patent and other issues. Existing products are available for jelly fish GFP and DsRed derived RFP fusions. Publications using such a limited number of camelid antibodies have been amazing so far—dozens in top journals within the last few months and after only a short period of time since product launch.
New Product of the Week 05-09-10 to 05-16-10: RFP-Trap for mCherry, mRFP1, mOrange, mPlum, and mRuby etc.
Promotion of the Week 05-09-10 to 05-16-10: Purchase our ThermoExp500 PCR Thermocycer for $4,650.00, and qualify for $200 off or for a $300 credit toward any other Allele Biotech product or service! http://www.allelebiotech.com/allele3/EQ.php
Original BioTechniques Article http://www.biotechniques.com/news/biotechniquesNews/biotechniques-257771.html?utm_source=BioTechniques+Newsletters+%2526+e-Alerts&utm_campaign=b94f127de0-Methods+Newsletter&utm_medium=email
Expanding the Camelid Antibody Product Line
While Chromotek GFP-Trap resin has become one of the best sellers from the Allele Biotech’ Camelid 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.
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%).
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.
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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
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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!
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