The Experts below are selected from a list of 5214 Experts worldwide ranked by ideXlab platform
Jarrod A Marto - One of the best experts on this subject based on the ideXlab platform.
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Tandem Affinity Purification and mass spectrometry tap ms for the analysis of protein complexes
Current protocols in protein science, 2019Co-Authors: Guillaume Adelmant, Brijesh K Garg, Maria Tavares, Joseph D Card, Jarrod A MartoAbstract:Affinity Purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for Tandem Affinity Purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
Spyros Artavanistsakonas - One of the best experts on this subject based on the ideXlab platform.
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analyzing protein complexes in drosophila with Tandem Affinity Purification mass spectrometry
Developmental Dynamics, 2005Co-Authors: Alexey Veraksa, Andreas Bauer, Spyros ArtavanistsakonasAbstract:We describe the application of Tandem Affinity Purification–mass spectrometry (TAP-MS) to the study of protein complexes in Drosophila. We have constructed vectors for inducible expression of TAP-tagged fusion proteins in Drosophila cultured cells and in vivo. Using these vectors, we tagged, as a paradigm, several components of the Notch signaling pathway, isolated protein complexes containing the baits and associated proteins from cells and embryos, and identified the subunits by liquid chromatography–Tandem mass spectrometry (LC-MS/MS). Several known interactions involving Notch pathway elements were confirmed, and many novel potential interactions were uncovered. For some of the novel associations, we validated the interaction genetically and biochemically. We conclude that TAP, in combination with MS, can be used as an effective method for the studies of the Drosophila proteome. Developmental Dynamics 232:827–834, 2005. © 2005 Wiley-Liss, Inc.
Guillaume Adelmant - One of the best experts on this subject based on the ideXlab platform.
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Tandem Affinity Purification and mass spectrometry tap ms for the analysis of protein complexes
Current protocols in protein science, 2019Co-Authors: Guillaume Adelmant, Brijesh K Garg, Maria Tavares, Joseph D Card, Jarrod A MartoAbstract:Affinity Purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for Tandem Affinity Purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
Jelle Van Leene - One of the best experts on this subject based on the ideXlab platform.
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isolation of protein complexes from the model legume medicago truncatula by Tandem Affinity Purification in hairy root cultures
Plant Journal, 2016Co-Authors: Jonas Goossens, Dominique Eeckhout, Alan Walton, Annick De Keyser, Nathan De Geyter, Jan Mertens, Jacob Pollier, Jennifer Fiallosjurado, Rebecca De Clercq, Jelle Van LeeneAbstract:Tandem Affinity Purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.
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an improved toolbox to unravel the plant cellular machinery by Tandem Affinity Purification of arabidopsis protein complexes
Nature Protocols, 2015Co-Authors: Jelle Van Leene, Dominique Eeckhout, Geert Persiau, Eveline Van De Slijke, Bernard Cannoot, Nancy De Winne, Leen Vercruysse, Maarten Dedecker, Aurine Verkest, Klaas VandepoeleAbstract:Tandem Affinity Purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double Affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-Tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.
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isolation of transcription factor complexes from arabidopsis cell suspension cultures by Tandem Affinity Purification
Methods of Molecular Biology, 2011Co-Authors: Jelle Van Leene, Erwin Witters, Dominique Eeckhout, Geert Persiau, Eveline Van De Slijke, Jan Geerinck, Gert Van Isterdael, Geert De JaegerAbstract:Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput Tandem Affinity Purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and Tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.
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boosting Tandem Affinity Purification of plant protein complexes
Trends in Plant Science, 2008Co-Authors: Jelle Van Leene, Erwin Witters, Dirk Inze, Geert De JaegerAbstract:Protein-interaction mapping based on the Tandem Affinity Purification (TAP) approach has been successfully established for several systems, such as yeast and mammalian cells. However, relatively few protein complex Purifications have been reported for plants. Here, we highlight solutions for the pitfalls and propose a major breakthrough in the quest for a better TAP tag in plants.
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a Tandem Affinity Purification based technology platform to study the cell cycle interactome in arabidopsis thaliana
Molecular & Cellular Proteomics, 2007Co-Authors: Jelle Van Leene, Hilde Stals, Dominique Eeckhout, Geert Persiau, Eveline Van De SlijkeAbstract:Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput Tandem Affinity Purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, Tandem Affinity Purification adapted for plant cells, matrix-assisted laser desorption ionization Tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.
Marius Ueffing - One of the best experts on this subject based on the ideXlab platform.
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strep flag Tandem Affinity Purification sf tap to study protein interactions
Current protocols in protein science, 2009Co-Authors: Christian Johannes Gloeckner, Karsten Boldt, Marius UeffingAbstract:In recent years, several methods have been developed to analyze protein-protein interactions under native conditions. One of them, Tandem Affinity Purification (TAP), combines two Affinity-Purification steps to allow isolation of high-purity protein complexes. This unit presents a methodological workflow based on an SF-TAP tag comprising a doublet Strep-tag II and a FLAG moiety optimized for rapid as well as efficient Tandem Affinity Purification of native proteins and protein complexes in higher eukaryotic cells. Depending on the stringency of Purification conditions, SF-TAP allows both the isolation of a single tagged-fusion protein of interest and Purification of protein complexes under native conditions.
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Tandem Affinity Purification of ciliopathy associated protein complexes
Methods in Cell Biology, 2009Co-Authors: Karsten Boldt, Marius Ueffing, Christian Johannes Gloeckner, Jeroen Van Reeuwijk, Ronald RoepmanAbstract:Ciliary dysfunction has recently been recognized as a cause for a growing number of genetically inherited disorders termed ciliopathies. Ciliopathy-associated proteins are organized in cell/context-specific complexes and in shared regulatory circuits in cilia of affected tissues. Thus, the identification of protein interactions involved in ciliary function provides a valid starting point to molecularly dissect normal ciliary function in a context and tissue specific fashion, identify novel functional candidate genes for ciliopathies as well as uncover the molecular defects that cause ciliary disease on the cellular level. Numerous methods have been developed over the years to categorize protein-protein interactions as well as to isolate native protein complexes. This chapter presents the details of an optimized Tandem Affinity Purification (TAP) procedure, employing a 4.6-kDa tag containing a doublet Strep-tag II and a FLAG octapeptide epitope tag. In contrast to other TAP methods, utilization of these two Affinity-binding moieties eliminates the need for a proteolytic cleavage step and allows the undisturbed isolation of the native protein complex binding to the tag-fusion protein. The small size of the synthetic and hydrophilic moieties of the Strep/FLAG TAP tag greatly reduce nonspecific protein binding as well as steric hindrance. We have employed this tag successfully for the identification of the lebercilin interactome, a ciliary and ciliopathy-associated protein network. Promising developments include quantitative proteomics (stable isotope labelling with amino acids in cell culture; SILAC) and BAC (bacterial artificial chromosome) recombineering to express tagged genes in higher eukaryotes, further expanding the versatility of this procedure.
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Tandem Affinity Purification of protein complexes from mammalian cells by the strep flag sf tap tag
Methods of Molecular Biology, 2009Co-Authors: Christian Johannes Gloeckner, Karsten Boldt, Annette Schumacher, Marius UeffingAbstract:Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the Tandem Affinity Purification (TAP) tag has enabled efficient and large-scale Purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward Purification of protein complexes from mammalian cells. It consists of a Tandem Strep-tag II and a FLAG epitope (SF-TAP). The SF-TAP tag allows a native elution of protein complexes without proteolytic cleavage needed in the original TAP procedure. Besides the SF-TAP protocol, the principal idea of a pathway mapping by subsequent tagging of copurified proteins is demonstrated for the interactome of the MAPKKK Raf.
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a novel Tandem Affinity Purification strategy for the efficient isolation and characterisation of native protein complexes
Proteomics, 2007Co-Authors: Christian Johannes Gloeckner, Karsten Boldt, Annette Schumacher, Ronald Roepman, Marius UeffingAbstract:Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the Tandem Affinity Purification (TAP) tag has enabled an efficient and large-scale Purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a Tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium Affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward Purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the Purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.
