VHH

Visualizing Endogenous Synaptic Proteins in Living Neurons

The recently published method is based on the generation of disulfide-free “intrabodies”, a structure from the 10th fibronectin type III domain known as FingRs. These affinity molecules were fused to GFP for direct fluorescence miscroscopy. The FingRs do not need di-sulfite bonds and are therefore better folders in mammalian cells. Specifically, a library was screened with in vitro display to identify FingRs that bind two synaptic proteins, Gephyrin and PSD95. After the initial selection, the researchers from USC secondarily screened binders using a cellular localization assay to identify potential FingRs that bind at high affinity in an intracellular environment. As it turned out, only 10-20% of the original positive clones bind well inside the cells, suggesting this type of further screening was a critical step.

The expression of intrabody is transcriptionally regulated by the target protein through a ZFN-repressor fusion. This transcriptional control system matches the expression of the intrabody to that of the target protein regardless of the target’s expression level. This design virtually eliminates unbound FingR, resulting in very low background that allows unobstructed visualization of the target proteins. As result, the FingRs presented in this study enabled live cell visualization of excitatory and inhibitory synapses, and apparently without affecting neuronal function.

Technically, the reason to use in vitro mRNA display was required by the need to use a large library (>10exp12, beyond the limit of the more commonly used phase display) to find good binders. A similar visualization system can be established using more potent affinity domains such as the VHH single-domain antibodies that have only one, sometimes dispensable, di-sulfite bond. The VHH domain nanobodies can be more easily isolated from camelid animals. Another improvement to the visualization system can be made by using stronger, superresolution-ready FPs such as mNeonGreen or mMaple to enable single molecule imaging, which is particularly interesting for studying synapses and applied to the BRAIN initiative.

Gross et al. Neuron, June 2013, http://www.ncbi.nlm.nih.gov/pubmed/23791193

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VHH Nanobodies in Superresolution 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 antibodies 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.

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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

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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.

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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

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Finding the Best Capture Reagents

As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

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