VHH Nanobodies in Super-Resolution Imaging and More

From the large number of recent publications using GFP-Trap beads, it appears that GFP-Trap is on the way to becoming one the most popular tags for co-IP thanks to its unparalleled “cleanness” of precipitated protein bands and its quantitative binding capabilities. As described previously, the antibody conjugated on the GFP-Trap beads is a single-domain antigen binding module from camelid single-chain antibodies. Termed VHH, this domain is only ~12 kD and can fit into structures that other types of antibodies cannot. We have successfully created VHH antibodies against a number of neural factors as a research project for the NIDA/NIH.

VHH proteins are often called nanobodies as a result of their size (1.5 – 2.5nm) and binding affinity ( GFP-trap has a binding affinity of 0.59nM). In addition to their use for co-IP, VHH antibodies have proven themselves as a resilient tool for various other applications. Anti-GFP nanobodies, for example, are currently used to enhance the fluorescence of GFP (GFP-trap booster utilizes the same VHH binding antibody coupled to a fluorescent dye); others have used VHH antibodies that can insert into certain part of GFP to dim the fluorescence signal . More recently, Ries et al. published in Nature Methods that the anti-GFP nanobodies offered a simple and versatile method for super-resolution imaging (i.e. PALM)-previously super-resolution imaging requires photoconvertible fluorescent proteins (such as Eos, mClavGR2). With dye-conjugated nanobodies, generating fusions to these newer FPs is no longer needed, however, using the nanobody super-imaging method requires fixing and permeabilizing the cells.

When using anti-GFP VHH reagents you need to be aware that other fluorescent proteins can also be recognized, if they were derived from the avGFP (jellyfish GFP). Also, some GFPs are not recognized if they are from another species, or engineered such as our mWasabi. We are producing newer and brighter GFP/YFPs based on the lancelet YFP protein to offer alternative series that will not be cross-recognized by the GFP-Trap antibodies.

Tags: Co-IP, GFP-Trap, Lancelet YFP, mClavGR2, mWasabi, nanobodies, PALM, RFP-Trap, VHH

Record number of papers citing the GFP-Trap group products in 2011

The following are references in regards to GFP Trap published in the second half of 2011 (not a complete list); a high quality GFP-binding protein based on a single domain antibody derived from Camelids. It is characterized by a small barrel shaped structure (13 KDa, 2.5nm X 4.5 nm) and a very high stability (stable up to 70°C, functional within 2M NaCl or 0.5% SDS). With much greater stability, specificity, and affnity, GFP-Trap®, the recent addition to antibodies for immunoprecipitation, should make GFP the most suitable tag for immunoprecipitation assays.

For live PubMed links, view this version please.

Krastev, D. B., Slabicki, M., et al. (2011). A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nature Cell Biology. 13: 809-818. PubMed

Aboobakar, E. F., Wang, X., et al. (2011). The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high temperature stress responses in Cryptococcus neoformans. Eukaryotic Cell. EC. 05148-05111v05141. PubMed

Uhrig, R. G. and Moorhead, G. B. G. (2011). Two ancient bacterial-like PPP family phosphatases from Arabidopsis thaliana are highly conserved plant proteins that possess unique properties. Plant Physiology. PubMed

Larance, M., Kirkwood, K. J., et al. (2011). Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway. Molecular & Cellular Proteomics. PubMed

Hattersley, N., Shen, L., et al. (2011). The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Molecular Biology of the Cell. 22: 78-90. PubMed

Rancz, E. A., Franks, K. M., et al. (2011). Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics. Nature Neuroscience. 14: 527-532. PubMed

Palmer, C. S., Osellame, L. D., et al. (2011). MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO reports. 12: 565-573. PubMed

Pichler, G., Wolf, P., et al. (2011). Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways. Journal of Cellular Biochemistry. 112: 2585-2593. PubMed

Mitchell, L., Lau, A., et al. (2011). Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4. PLoS One. 6: e25336. PubMed

Engeland, C. E., Oberwinkler, H., et al. (2011). The cellular protein Lyric interacts with HIV-1 Gag. Journal of virology. JVI. 00174-00111v00171. PubMed

Wang, C. and Youle, R. (2011). Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1′s inhibitory effect on Bak. Oncogene. PubMed

Tulloch, L. B., Howie, J., et al. (2011). The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation. Journal of Biological Chemistry. 286: 36020-36031. PubMed

Sun, L. and Wang, C. C. (2011). The Structural Basis of Localizing Polo-Like Kinase to the Flagellum Attachment Zone in Trypanosoma brucei. PLoS One. 6: e27303. PubMed

Bouttier, M., Saumet, A., et al. (2011). Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation. Nucleic acids research. PubMed

Matos, J., Blanco, M. G., et al. (2011). Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis. Cell. 147: 158-172. PubMed

Nagel, C. H., Albrecht, N., et al. (2011). Herpes Simplex Virus Immediate-Early Protein ICP0 Is Targeted by SIAH-1 for Proteasomal Degradation. Journal of virology. 85: 7644. PubMed

Studencka, M., Konzer, A., et al. (2011). Novel roles of C. elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression. Molecular and Cellular Biology.PubMed

Muehlen, S., Ruchaud-Sparagano, M. H., et al. (2011). Proteasome-independent Degradation of Canonical NFŒ?B Complex Components by the NleC Protein of Pathogenic Escherichia coli. Journal of Biological Chemistry. 286: 5100. PubMed

Galan, J. A., Paris, L. L., et al. (2011). Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap. Journal of the American Society for Mass Spectrometry. 1-10. PubMed

Chamousset, D., De Wever, V., et al. (2010). RRP1B Targets PP1 to Mammalian Cell Nucleoli and is Associated with Pre-60S Ribosomal Subunits. Mol Biol Cell. PubMed

Kovacs, E. M., Verma, S., et al. (2011). N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nature Cell Biology. 13: 934-943. PubMed

Boysen, K. E. and Matuschewski, K. (2011). Arrested oocyst maturation in Plasmodium parasites lacking type II NADH: ubiquinone dehydrogenase. Journal of Biological Chemistry. 286: 32661-32671. PubMed

Mortusewicz, O., Fouquerel, E., et al. (2011). PARG is recruited to DNA damage sites through poly (ADP-ribose)-and PCNA-dependent mechanisms. Nucleic acids research. 39: 5045. PubMed

Graewe, S., Rankin, K. E., et al. (2011). Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathogens. 7: e1002224. PubMed

Yang, X. D., Huang, S., et al. (2011). Distinct and mutually inhibitory binding by two divergent Œ?-catenins coordinates TCF levels and activity in C. elegans. Development. 138: 4255-4265. PubMed

Pollithy, A., Romer, T., et al. (2011). Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense. Applied and environmental microbiology. 77: 6165-6171. PubMed

Kozubowski, L., Thompson, J. W., et al. (2011). Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics. PLoS One. 6: e25280. PubMed

Garcia-Gomez, J. J., Lebaron, S., et al. (2011). Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae. Molecular and Cellular Biology. 31: 4156-4164. PubMed

Van Damme, D., Gadeyne, A., et al. (2011). Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proceedings of the National Academy of Sciences. 108: 615. PubMed

Qvist, P., Huertas, P., et al. (2011). CtIP Mutations Cause Seckel and Jawad Syndromes. PLoS Genetics. 7: e1002310. PubMed

Labella, S., Woglar, A., et al. (2011). Polo Kinases Establish Links between Meiotic Chromosomes and Cytoskeletal Forces Essential for Homolog Pairing. Developmental Cell. PubMed

Harterink, M., Port, F., et al. (2011). A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biology. 13: 914-923. PubMed

Konopacki, F. A., Jaafari, N., et al. (2011). Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis. Proceedings of the National Academy of Sciences.PubMed

Chuhma, N., Tanaka, K. F., et al. (2011). Functional connectome of the striatal medium spiny neuron. The Journal of Neuroscience. 31: 1183-1192. PubMed

Jackson, B. R., Boyne, J. R., et al. (2011). An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export. PLoS Pathogens. 7: e1002138. PubMed

Tags: anti-GFP, camelid antibodies, Camelid Antibodies, Nanobodies, VHH, Chromotek, Co-IP, GFP-Trap, GFPTrap, immunoprecipitation, pulldown, RFPTrap, VHH

Top 10 List of Most Viewed AlleleBlogs in 2011

The ballot is in—among the “usual suspect” hot topics, iPS takes the top honor and most entries; Camelid antibodies, although not really presented as a typical AlleleBlog in 2011, made it to the top 3. shRNA cloning and RNAi screening are still on a lot of people’s minds, so it seems.

Method: total visits to each blog since our new webpage was launched in July was counted.

1) Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming
http://blog.allelebiotech.com/2011/10/fusion-of-the-transcription-domain-to-ips-factors-radically-enhances-reprogramming/

2) Methods of iPSC Generation Update
http://blog.allelebiotech.com/2011/08/methods-of-ipsc-generation-update/

3) About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011
http://blog.allelebiotech.com/2011/09/about-50-papers-cited-the-use-of-gfp-trap-camelid-antibody-so-far-in-2011/

4) Big Potential in Using Protozoans for Producing Mammalian Proteins
http://blog.allelebiotech.com/2011/09/big-potential-in-using-protozoans-for-producing-mammalian-proteins/

5) How do you make shRNA-expressing viruses for function screening?
http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

6) Creating ground-state human iPSCs
http://blog.allelebiotech.com/2011/10/creating-ground-state-human-ipsc/

7) Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
http://blog.allelebiotech.com/2011/03/recombinase-mediated-cassette-exchange-rmce-and-integrase-swappable-in-vivo-targeting-element-insite/

Development of Cell Lines from iPSCs for Bioassays
http://blog.allelebiotech.com/2011/11/development-of-cell-lines-from-ipscs-for-bioassays/

9) Choosing siRNA, shRNA, and miRNA for Gene Silencing
blog.allelebiotech.com/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/

10) Allele Biotech’s Box Swap Program
http://blog.allelebiotech.com/2009/07/allele-biotechs-box-swap-program/

Have a successful 2012!

Tags: camelid antibodies, environment friendly research, GFP-Trap, iPS, mRNA iPS, RiPSC, RNAi screening, shRNA lentivirus packaging

About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011

With their ability to quantitatively pulldown GFP-tagged proteins, GFP-Trap (or RFP-Trap for DsRed-derived fluorescent proteins) beads have gained ground in becoming the reagent of choice for immuno-coprecipitation. The complexes isolated from GFP-Trap agarose or magnetic beads can be easily analyzed without interference from light or heavy IgG chains typically present after monoclonal or polyclonal antibody precipitation. Since the market launch of GFP-Trap, in each of the past 3 years, the number of publications citing GFP-Trap more has than doubled and there is no sign of that rate slowing down any time soon.

In 2011 alone, 48 research groups have published their results with data generated through use of GFP-Trap (not including other related products such as GFP-Booster, GFP-MultiTrap). Research topics in these recent publications include identification of domains of the zinc finger protein 638 (ZNF638) that interacts with C/EBPb when promoting adipocyte differentiation [1]; identification of phosphorylation site on Cdc42-associated kinase (Ack) by LC-MS/MS after immunoprecipitation [2]; and analysis of the activities of myosin heavy-chain kinases (MHCKs) in wild-type vs Htt mutant Dictyostelium discoideum, a cellular model for studying the Huntingon disease [3].

The use of GFP-Trap beads is a simple bind-wash-elute procedure that involves just one antibody already immobilized on either agarose or magnetic beads. Camelid antibodies, especially their VHH single domain fragments such as those used in GFP-Trap or RFP-Trap, are very stable (they can be shipped and temporarily stored at room temperature). The consistency of performance is very high; as a matter of fact, this line of products requires the lowest amount of technical support among all of our products. If you are still using tags like FLAG, V5, HA, etc., you should consider trying GFP as both a fluorescence and co-IP tag in your future experiments for obtaining results you previously could not obtain.

New Product of the Week: Non-Integrating iPSC Generation Kits. First of its kind on the market. Click to read more about mRNA-based reprogramming.

Promotion of the Week: Save 15% to save the environment by using EcoCulture Dishes at 30% less plastic for better imaging. Code: 091911DISH when call or email us.

Blog References:
[1] Meruvu, S. et al. “Regulation of Adipocyte Differentiation by the Zinc Finger Protein ZNF638″ JBC 2011
[2] Shen, H. et al. “Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin” Molecular Biology of the Cell 2011
[3] Wang, Y. et al. “Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation” Molecular Biology of the Cell 2011

Tags: anti-GFP, camelid antibodies, Camelid Antibodies, Nanobodies, VHH, Chromotek, Co-IP, GFP-Trap, GFPTrap, immunoprecipitation, pulldown, RFPTrap, VHH

Updated Publication List Using GFP-Trap Related Products

2011

Courtesey: list prepared by ChromoTek

Kastner, P. M., Schleicher, M., et al. (2011). The NDR Family Kinase NdrA of Dictyostelium Localizes to the Centrosome and Is Required for Efficient Phagocytosis. Traffic. 12: 301-312.

Guizetti, J., Schermelleh, L., et al. (2011). Cortical Constriction During Abscission Involves Helices of ESCRT-III-Dependent Filaments. Science.

Muhlen, S., Ruchaud-Sparagano, M. H., et al. (2011). Proteasome-independent Degradation of Canonical NF{kappa}B Complex Components by the NleC Protein of Pathogenic Escherichia coli. J Biol Chem. 286: 5100-5107.

Speck, J., Arndt, K. M., et al. (2011). Efficient phage display of intracellularly folded proteins mediated by the TAT pathway. Protein Eng Des Sel.

Heinrich, C., Gascon, S., et al. (2011). Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat Protoc. 6: 214-228.

Qin, W., Leonhardt, H., et al. (2011). Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1. J Cell Biochem. 112: 439-444.

Shen, H., Ferguson, S. M., et al. (2011). Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin. Mol Biol Cell. 22: 493-502.

Wilkinson, K. A. and Henley, J. M. (2011). Analysis of metabotropic glutamate receptor 7 as a potential substrate for SUMOylation. Neurosci Lett.

Reininger, L., Wilkes, J. M., et al. (2011). An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony. Mol Microbiol. 79: 205-221.

Bubeck, D., Reijns, M. A., et al. (2011). PCNA directs type 2 RNase H activity on DNA replication and repair substrates. Nucleic Acids Res.

Dissanayake, K., Toth, R., et al. (2011). ERK/p90(RSK)/14-3-3 signalling has an impact on expression of PEA3 Ets transcription factors via the transcriptional repressor capicua. Biochem J. 433: 515-525.

Kuipers, M. A., Stasevich, T. J., et al. (2011). Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload. J Cell Biol. 192: 29-41.

Frauer, C., Rottach, A., et al. (2011). Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1. PLoS One. 6: e16627.

2010

Paris, L. L., Hu, J., et al. (2010). Regulation of Syk by phosphorylation on serine in the linker insert. J Biol Chem. 285: 39844-39854.

Korzeniowski, M. K., Manjarres, I. M., et al. (2010). Activation of STIM1-Orai1 involves an intramolecular switching mechanism. Sci Signal. 3: ra82.

Chamousset, D., De Wever, V., et al. (2010). RRP1B Targets PP1 to Mammalian Cell Nucleoli and is Associated with Pre-60S Ribosomal Subunits. Mol Biol Cell.

Thorslund, T., McIlwraith, M. J., et al. (2010). The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat Struct Mol Biol. 17: 1263-1265.

Erdel, F., Schubert, T., et al. (2010). Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites. Proc Natl Acad Sci U S A.

Geoffroy, M. C., Jaffray, E. G., et al. (2010). Arsenic-induced, SUMO-dependent Recruitment of RNF4 into PML Nuclear Bodies. Mol Biol Cell. (PudMed)

Boulon, S., Pradet-Balade, B., et al. (2010). HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol Cell. 39: 912-924.

Schmitz, K. M., Mayer, C., et al. (2010). Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev. 24: 2264-2269.

Bakondi, B. and Spees, J. L. (2010). Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice. Biochem Biophys Res Commun. 400: 212-218.

Vermeulen, M., Eberl, H. C., et al. (2010). Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell. 142: 967-980.

Pozo-Guisado, E., Campbell, D. G., et al. (2010). Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry. J Cell Sci. 123: 3084-3093.

Kaidi, A., Weinert, B. T., et al. (2010). Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science. 329: 1348-1353.

Dzamko N., et al. (2010). Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 430: 405-413.

Nichols R. J., et al. (2010). 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson’s disease-associated mutations and regulates cytoplasmic localization. Biochem J 430: 393-404.

Polo S. E., et al. (2010). Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4. EMBO J.

Babiano R., et al. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 38: 5177-5192.

Loiseau P., et al. (2010). Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility. Development 137: 2763-2772.

Dubin, M., Fuchs, J., et al. (2010). Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis. Nucleic Acids Res.

Pabis, M., Neufeld, N., et al. (2010). Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20. Nucleus. 1: 412-421.

Van Dessel N., et al. (2010). The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets. Nucleic Acids Res.

Rass U., et al. (2010). Mechanism of Holliday junction resolution by the human GEN1 protein. Genes Dev 24: 1559-1569.

MacKay C., et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142: 65-76.

Ommen G., et al. (2010). The co-chaperone SGT of Leishmania donovani is essential for the parasite’s viability. Cell Stress Chaperones 15: 443-455.

Fulcher A. J., et al. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem 285: 17744-17753.

Taniue K., et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development 137: 1755-1764.

Kovanich, D., van der Heyden, M. A., et al. (2010). Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase. Chembiochem. 11: 963-971.

Boulon S., 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: 861-879.

Slabicki M., et al. (2010). A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 8: e1000408.

Laxman, S., Sutter, B. M., et al. (2010). Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters. PLoS One. 5: e12595.

Bergbauer, M., Kalla, M., et al. (2010). CpG-methylation regulates a class of Epstein-Barr virus promoters. PLoS Pathog. 6.

Kalla M., et al. (2010). AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome. Proc Natl Acad Sci U S A 107: 850-855.

Bellanger S., et al. (2010). The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J Virol 84: 437-444.

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Promotion of the week 032111-032711: 10% off on all mWasabi and mTFP1 organelle marker fusion vectors. Email abbashussain@allelebiotech.com and include the code FUSION to redeem this offer.

Tags: anti-GFP, anti-RFP, camelid antibodies, Chromotek, GFP booster, GFP-Trap, Multi-Trap, single domain antibodies